Method for making multispecific antibodies having heteromultimeric and common components

ABSTRACT

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method further involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and/or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 09/520,130, filed Mar. 7, 2000, which is a continuation applicationof U.S. application Ser. No. 09/070,416, filed Apr. 30, 1998, claimingpriority under 35 USC 119(e) to provisional application No. 60/050,661,filed Jun. 24, 1997, and provisional application No. 60/046,816, filedMay 2, 1997, which applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a method for making multispecific antibodieshaving heteromultimeric heavy chain components and common light chaincomponents such as bispecific antibodies, bispecific immunoadhesins, aswell as antibody-immunoadhesin chimeras and the heteromultimericpolypeptides made using the method.

BACKGROUND OF THE INVENTION

Bispecific Antibodies

Bispecific antibodies (BsAbs) which have binding specificities for atleast two different antigens have significant potential in a wide rangeof clinical applications as targeting agents for in vitro and in vivoimmunodiagnosis and therapy, and for diagnostic immunoassays.

In the diagnostic areas, bispecific antibodies have been very useful inprobing the functional properties of cell surface molecules and indefining the ability of the different Fc receptors to mediatecytotoxicity (Fanger et al., Crit. Rev. Immunol. 12:101-124 (1992)).Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990) describe otherdiagnostic applications for BsAbs. In particular, BsAbs can beconstructed to immobilize enzymes for use in enzyme immunoassays. Toachieve this, one arm of the BsAb can be designed to bind to a specificepitope on the enzyme so that binding does not cause enzyme inhibition,the other arm of the BsAb binds to the immobilizing matrix ensuring ahigh enzyme density at the desired site. Examples of such diagnosticBsAbs include the rabbit anti-IgG/anti-ferritin BsAb described byHammerling et al., J. Exp. Med. 128:1461-1473 (1968) which was used tolocate surface antigens. BsAbs having binding specificities for horseradish peroxidase (HRP) as well as a hormone have also been developed.Another potential immunochemical application for BsAbs involves theiruse in two-site immunoassays. For example, two BsAbs are producedbinding to two separate epitopes on the analyte protein—one BsAb bindsthe complex to an insoluble matrix, the other binds an indicator enzyme(see Nolan et al., supra).

Bispecific antibodies can also be used for in vitro or in vivoimmunodiagnosis of various diseases such as cancer (Songsivilai et al.,Clin. Exp. Immunol. 79:315 (1990)). To facilitate this diagnostic use ofthe BsAb, one arm of the BsAb can bind a tumor associated antigen andthe other arm can bind a detectable marker such as a chelator whichtightly binds a radionuclide. Using this approach, Le Doussal et al.made a BsAb useful for radioimmunodetection of colorectal and thyroidcarcinomas which had one arm which bound a carcinoembryonic antigen(CEA) and another arm which bound diethylenetriaminepentacetic acid(DPTA). See Le Doussal et al., Int. J. Cancer Suppl. 7:58-62 (1992) andLe Doussal et al., J. Nucl. Med. 34:1662-1671 (1993). Stickney et al.similarly describe a strategy for detecting colorectal cancersexpressing CEA using radioimmunodetection. These investigators describea BsAb which binds CEA as well as hydroxyethylthiourea-benzyl-EDTA(EOTUBE). See Stickney et al., Cancer Res. 51:6650-6655 (1991).

Bispecific antibodies can also be used for human therapy in redirectedcytotoxicity by providing one arm which binds a target (e.g. pathogen ortumor cell) and another arm which binds a cytotoxic trigger molecule,such as the T-cell receptor or the Fcγ receptor. Accordingly, bispecificantibodies can be used to direct a patient's cellular immune defensemechanisms specifically to the tumor cell or infectious agent. Usingthis strategy, it has been demonstrated that bispecific antibodies whichbind to the FcγRIII (i.e. CD16) can mediate tumor cell killing bynatural killer (NK) cell/large granular lymphocyte (LGL) cells in vitroand are effective in preventing tumor growth in vivo. Segal et al.,Chem. Immunol. 47:179 (1989) and Segal et al., Biologic Therapy ofCancer 2(4) DeVita et al. eds. J. B. Lippincott, Philadelphia (1992)p. 1. Similarly, a bispecific antibody having one arm which bindsFcγRIII and another which binds to the HER2 receptor has been developedfor therapy of ovarian and breast tumors that overexpress the HER2antigen. (Hseih-Ma et al. Cancer Research 52:6832-6839 (1992) and Weineret al. Cancer Research 53:94-100 (1993)). Bispecific antibodies can alsomediate killing by T cells. Normally, the bispecific antibodies link theCD3 complex on T cells to a tumor-associated antigen. A fully humanizedF(ab′)₂ BsAb consisting of anti-CD3 linked to anti-p185^(HER2) has beenused to target T cells to kill tumor cells overexpressing the HER2receptor. Shalaby et al., J. Exp. Med. 175(1):217 (1992). Bispecificantibodies have been tested in several early phase clinical trials withencouraging results. In one trial, 12 patients with lung, ovarian orbreast cancer were treated with infusions of activated T-lymphocytestargeted with an anti-CD3/anti-tumor (MOC31) bispecific antibody. deLeijet al. Bispecific Antibodies and Targeted Cellular Cytotoxicity,Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991) p. 249. Thetargeted cells induced considerable local lysis of tumor cells, a mildinflammatory reaction, but no toxic side effects or anti-mouse antibodyresponses. In a very preliminary trial of an anti-CD3/anti-CD19bispecific antibody in a patient with B-cell malignancy, significantreduction in peripheral tumor cell counts was also achieved. Clark etal. Bispecific Antibodies and Targeted Cellular Cytotoxicity,Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991) p. 243. See alsoKroesen et al., Cancer Immunol. Immunother. 37:400-407 (1993), Kroesenet al., Br. J. Cancer 70:652-661 (1994) and Weiner et al., J. Immunol.152:2385 (1994) concerning therapeutic applications for BsAbs.

Bispecific antibodies may also be used as fibrinolytic agents or vaccineadjuvants. Furthermore, these antibodies may be used in the treatment ofinfectious diseases (e.g. for targeting of effector cells to virallyinfected cells such as HIV or influenza virus or protozoa such asToxoplasma gondii), used to deliver immunotoxins to tumor cells, ortarget immune complexes to cell surface receptors (see Fanger et al.,supra).

Use of BsAbs has been effectively hindered by the difficulty ofobtaining BsAbs in sufficient quantity and purity. Traditionally,bispecific antibodies were made using hybrid-hybridoma technology(Millstein and Cuello, Nature 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules, of which only one has the correct bispecific structure (seeFIG. 1A). The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. See, for example, (Smith, W., et al. (1992)Hybridoma 4:87-98; and Massimo, Y. S., et al. (1997) J. Immunol. Methods201:57-66). Accordingly, techniques for the production of greater yieldsof BsAb have been developed. To achieve chemical coupling of antibodyfragments, Brennan et al., Science 229:81 (1985) describe a procedurewherein intact antibodies are proteolytically cleaved to generateF(ab′)₂ fragments. These fragments are reduced in the presence of thedithiol complexing agent sodium arsenite to stabilize vicinal dithiolsand prevent intermolecular disulfide formation. The Fab′ fragmentsgenerated are then converted to thionitrobenzoate (TNB) derivatives. Oneof the Fab′-TNB derivatives is then reconverted to the Fab′-thiol byreduction with mercaptoethylamine and is mixed with an equimolar amountof the other Fab′-TNB derivative to form the BsAb. The BsAbs producedcan be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli. which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe theproduction of a fully humanized BsAb F(ab′)₂ molecule having one armwhich binds p185^(HER2) and another arm which binds CD3. Each Fab′fragment was separately secreted from E. coli. and subjected to directedchemical coupling in vitro to form the BsAb. The BsAb thus formed wasable to bind to cells overexpressing the HER2 receptor and normal humanT cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets. See also Rodrigues etal., Int. J. Cancers (Suppl.) 7:45-50 (1992).

Various techniques for making and isolating BsAb fragments directly fromrecombinant cell cultures have also been described. For example,bispecific F(ab′)₂ heterodimers have been produced using leucine zippers(Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of anti-CD3 and anti-interleukin-2 receptor (IL-2R) antibodiesby gene fusion. The antibody homodimers were reduced at the hinge regionto form monomers and then reoxidized to form the antibody heterodimers.The BsAbs were found to be highly effective in recruiting cytotoxic Tcells to lyse HuT-102 cells in vitro. The advent of the “diabody”technology described by Hollinger et al., PNAS (USA) 90:6444-6448 (1993)has provided an alternative mechanism for making BsAb fragments. Thefragments comprise a heavy chain variable domain (V_(H)) connected to alight chain variable domain (V_(L)) by a linker which is too short toallow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for making BsAbfragments by the use of single chain Fv (sFv) dimers has also beenreported. See Gruber et al. J. Immunol. 152: 5368 (1994). Theseresearchers designed an antibody which comprised the V_(H) and V_(L)domains of an antibody directed against the T cell receptor joined by a25 amino acid residue linker to the V_(H) and V_(L) domains of ananti-fluorescein antibody. The refolded molecule bound to fluoresceinand the T cell receptor and redirected the lysis of human tumor cellsthat had fluorescein covalently linked to their surface.

It is apparent that several techniques for making bispecific antibodyfragments which can be recovered directly from recombinant cell culturehave been reported. However, full length BsAbs may be preferable to BsAbfragments for many clinical applications because of their likely longerserum half-life and possible effector functions.

Immunoadhesins

Immunoadhesins (Ia's) are antibody-like molecules which combine thebinding domain of a protein such as a cell-surface receptor or a ligand(an “adhesin”) with the effector functions of an immunoglobulin constantdomain. Immunoadhesins can possess many of the valuable chemical andbiological properties of human antibodies. Since immunoadhesins can beconstructed from a human protein sequence with a desired specificitylinked to an appropriate human immunoglobulin hinge and constant domain(Fc) sequence, the binding specificity of interest can be achieved usingentirely human components. Such immunoadhesins are minimally immunogenicto the patient, and are safe for chronic or repeated use.

Immunoadhesins reported in the literature include fusions of the T cellreceptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940(1987)); CD4 (Capon et al., Nature 337:525-531 (1989); Traunecker etal., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin or homing receptor (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); NPreceptors (Bennett et al., J. Biol. Chem. 266:23060-23067 (1991));inteferon γ receptor (Kurschner et al., J. Biol. Chem. 267:9354-9360(1992)); 4-1BB (Chalupny et al., PNAS (USA) 89:10360-10364 (1992)) andIgE receptor α (Ridgway and Gorman, J. Cell. Biol. Vol. 115, AbstractNo. 1448 (1991)).

Examples of immunoadhesins which have been described for therapeutic useinclude the CD4-IgG immunoadhesin for blocking the binding of HIV tocell-surface CD4. Data obtained from Phase I clinical trials in whichCD4-IgG was administered to pregnant women just before delivery suggeststhat this immunoadhesin may be useful in the prevention ofmaternal-fetal transfer of HIV. Ashkenazi et al., Intern. Rev. Immunol.10:219-227 (1993). An immunoadhesin which binds tumor necrosis factor(TNF) has also been developed. TNF is a proinflammatory cytokine whichhas been shown to be a major mediator of septic shock. Based on a mousemodel of septic shock, a TNF receptor immunoadhesin has shown promise asa candidate for clinical use in treating septic shock (Ashkenazi et al.,supra). Immunoadhesins also have non-therapeutic uses. For example, theL-selectin receptor immunoadhesin was used as an reagent forhistochemical staining of peripheral lymph node high endothelial venules(HEV). This reagent was also used to isolate and characterize theL-selectin ligand (Ashkenazi et al., supra).

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. Immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin. Binding studies indicated that the bispecific immunoglobulinfusion protein so formed had an enhanced ability to bind to a myeloidcell line compared to the monospecific immunoadhesins from which it wasderived.

Antibody-Immunoadhesin Chimeras

Antibody-immunoadhesin (Ab/Ia) chimeras have also been described in theliterature. These molecules combine the binding region of animmunoadhesin with the binding domain of an antibody.

Berg et al., PNAS (USA) 88:4723-4727 (1991) made a bispecificantibody-immunoadhesin chimera which was derived from murine CD4-IgG.These workers constructed a tetrameric molecule having two arms. One armwas composed of CD4 fused with an antibody heavy-chain constant domainalong with a CD4 fusion with an antibody light-chain constant domain.The other arm was composed of a complete heavy-chain of an anti-CD3antibody along with a complete light-chain of the same antibody. Byvirtue of the CD4-IgG arm, this bispecific molecule binds to CD3 on thesurface of cytotoxic T cells. The juxtaposition of the cytotoxic cellsand HIV-infected cells results in specific killing of the latter cells.

While Berg et al. supra describe a bispecific molecule that wastetrameric in structure, it is possible to produce a trimeric hybridmolecule that contains only one CD4-IgG fusion. See Chamow et al., J.Immunol. 153:4268 (1994). The first arm of this construct is formed by ahumanized anti-CD3 κ light chain and a humanized anti-CD3 γ heavy chain.The second arm is a CD4-IgG immunoadhesin which combines part of theextracellular domain of CD4 responsible for gp120 binding with the Fcdomain of IgG. The resultant Ab/Ia chimera mediated killing ofHIV-infected cells using either pure cytotoxic T cell preparations orwhole peripheral blood lymphocyte (PBL) fractions that additionallyincluded Fc receptor-bearing large granular lymphocyte effector cells.

In the manufacture of the multispecific antibody heteromultimers, it isdesirable to increase the yields of the desired heteromultimer over thehomomultimer(s). The current method of choice for obtainingFc-containing BsAb remains the hybrid hybridoma, in which two antibodiesare coexpressed (Milstein and Cuello, Nature 305:537-540 (1983)).

In hybrid hybridomas, heavy (H) chains typically form homodimers as wellas the desired heterodimers. Additionally, light (L) chains frequentlymispair with non-cognate heavy chains. Hence, coexpression of twoantibodies may produce up to ten heavy and light chain pairings (Suresh,M. R., et al. Methods Enzymol. 121:210-228 (1986)). These unwanted chainpairings compromise the yield of the BsAb and inevitably imposesignificant, and sometimes insurmountable, purification challenges(Smith, et al. (1992) supra; and Massimo, et al. (1997) supra).

Antibody heavy chains have previously been engineered to driveheterodimerization by introducing sterically complementary mutations inmultimerization domains at the C_(H)3 domain interface (Ridgway et al.Protein Eng. 9:617-621 (1996)) and optimization by phage display asdescribed herein. Chains containing the modified C_(H)3 domains yield upto approximately 90% heterodimer as judged by formation of anantibody/immunoadhesin hybrid (Ab/Ia). Heterodimerized heavy chains maystill mispair with the non-cognate light chain, thus hampering recoveryof the BsAb of interest.

SUMMARY OF THE INVENTION

This application describes a strategy which serves to enhance theformation of a desired heteromultimeric bispecific antibody from amixture of monomers by engineering an interface between a first andsecond polypeptide for hetero-oligomerization and by providing a commonvariable light chain to interact with each of the heteromeric variableheavy chain regions of the bispecific antibody. There are three possiblehetero- and homomultimers that can form from a first and secondpolypeptide, each of which is, in turn, associated with a first andsecond light chain, respectively. This gives rise to a total of tenpossible chain pairings (FIG. 1A). A method of enhancing the formationof the desired heteromultimer can greatly enhance the yield overundesired heteromultimers and homomultimers.

The preferred interface between a first and second polypeptide of theheteromultimeric antibody comprises at least a part of the C_(H)3 domainof an antibody constant domain. The domain of each of the first andsecond polypeptides that interacts at the interface is called themultimerization domain. Preferably, the multimerization domain promotesinteraction between a specific first polypeptide and a secondpolypeptide, thereby increasing the yield of desired heteromultimer(FIG. 1B). Interaction may be promoted at the interface by the formationof protuberance-into-cavity complementary regions; the formation ofnon-naturally occurring disulfide bonds; leucine zipper; hydrophobicregions; and hydrophilic regions. “Protuberances” are constructed byreplacing small amino acid side chains from the interface of the firstpolypeptide with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to theprotuberances are optionally created on the interface of the secondpolypeptide by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). Where a suitably positioned and dimensionedprotuberance or cavity exists at the interface of either the first orsecond polypeptide, it is only necessary to engineer a correspondingcavity or protuberance, respectively, at the adjacent interface.Non-naturally occurring disulfide bonds are constructed by replacing onthe first polypeptide a naturally occurring amino acid with a freethiol-containing residue, such as cysteine, such that the free thiolinteracts with another free thiol-containing residue on the secondpolypeptide such that a disulfide bond is formed between the first andsecond polypeptides (FIG. 1B).

Single chain Fv fragments from a large non-immunized phage displaylibrary (Vaughan, T. J. et al. (1996) Nature Biotechnology 14:309-314,herein incorporated by reference in its entirety) revealed V-gene usagein which V_(H) and V_(L) sequences derived from certain germline V-genesegments predominated. families predominated in the repertoire. Examplesof chain promiscuity in the repertoire were noted in which a particularheavy or light chain is found in combination with different partnerchains (Vaughan, T. J. et al. (1996) supra).

It is disclosed herein that the preparation of a desiredheteromultimeric multispecific antibody is enhanced when a common lightchain is provided to pair with each of the variable heavy chains of themultispecific antibody. Use of a common variable light chain reduces thenumber of monomers that must correctly pair to form the antigen bindingdomains by limiting the number of light chains from two or more lightchains (in a bispecific or multispecific antibody, respectively, priorto disclosure of the instant invention) to one light chain (in amultispecific antibody of the invention, see FIG. 1C).

Accordingly, the invention relates to a method of preparing aheteromultimeric multispecific antibody, the antibody comprising 1) afirst polypeptide and a second polypeptide (and additional polypeptidesaccord to the multiplicity of the antibody) which meet at an interface,wherein the first and additional polypeptides (i.e., a first and secondpolypeptide) each include a multimerization domain forming an interfacebetween the first and second (or at least one additional) polypeptides,and the multimerization domains promote stable interaction between firstand additional polypeptides, and 2) a binding domain in each of thefirst and at least one additional polypeptide (i.e. a secondpolypeptide), each binding domain comprising a variable heavy chain anda variable light chain, wherein the variable light chain of the firstpolypeptide and the variable light chain of the second polypeptide havea common amino acid sequence, which common sequence has an amino acidsequence identity to an original light chain of each of the polypeptidesof at least 80%, preferably at least 90%, more preferably at least 95%and most preferably 100% sequence identity. The method comprises thesteps of

(i) culturing a host cell comprising nucleic acid encoding the firstpolypeptide, the second polypeptide, and the common light chain whereinthe culturing is such that the nucleic acid is expressed; and

(ii) recovering the multispecific antibody from the host cell culture;

In a related embodiment of the invention the nucleic acid encoding thefirst polypeptide or the nucleic acid encoding the second polypeptide,or both, has been altered from the original nucleic acid to encode theinterface or a portion thereof.

In another embodiment of the method, the interface of the firstpolypeptide comprises a free thiol-containing residue which ispositioned to interact with a free thiol-containing residue of theinterface of the second polypeptide such that a disulfide bond is formedbetween the first and second polypeptides. According to the invention,the nucleic acid encoding the first polypeptide has been altered fromthe original nucleic acid to encode the free thiol-containing residue orthe nucleic acid encoding the second polypeptide has been altered fromthe original nucleic acid to encode the free thiol-containing residue,or both.

In another embodiment of the method, the nucleic acid encoding both thefirst polypeptide and at least one additional polypeptide (i.e., asecond polypeptide) are altered to encode the protuberance and cavity,respectively. Preferably the first and second polypeptides each comprisean antibody constant domain such as the C_(H)3 domain of a human IgG₁.

In another aspect, the invention provides a heteromultimer (such as abispecific antibody, bispecific immunoadhesin or antibody/immunoadhesinchimera) comprising a first polypeptide and a second polypeptide whichmeet at an interface. The interface of the first polypeptide comprises amultimerization domain which is positioned to interact with amultimerization domain on the at least one additional polypeptide (i.e.,a second polypeptide) to form an interface between the first and secondpolypeptide. In preferred embodiments of the invention, themultimerization domains are altered to promote interaction between aspecific first polypeptide and a specific second polypeptide, whichalterations include, but are not limited to, the generation of aprotuberance or cavity, or both; the generation of non-naturallyoccurring disulfide bonds; the generation of complementary hydrophobicregions; and the generation of complementary hydrophilic regions. Theheteromultimeric multispecific antibody may be provided in the form of acomposition further comprising a pharmaceutically acceptable carrier.

The invention also relates to a host cell comprising nucleic acidencoding the heteromultimeric multispecific antibody of the precedingparagraph wherein the nucleic acid encoding the first polypeptide and atleast one additional polypeptide (i.e., a second polypeptide) is presentin a single vector or in separate vectors. The host cell can be used ina method of making a heteromultimeric multispecific antibody whichinvolves culturing the host cell so that the nucleic acid is expressed,and recovering the heteromultimeric antibody from the cell culture.

In yet a further aspect, the invention provides a method of preparing aheteromultimeric multispecific antibody comprising:

(a) selecting a first nucleic acid encoding a first polypeptidecomprising an amino acid residue in the interface of the firstpolypeptide that is positioned to interact with an amino acid residue ofinterface of at least one additional polypeptide. In an embodiment thenucleic acid is altered from the original to encode the interactingamino acid residues. In another embodiment, the first nucleic acid isaltered to encode an amino acid residue having a larger side chainvolume, thereby generating a protuberance on the first polypeptide;

(b) altering a second nucleic acid encoding a second polypeptide so thatan amino acid residue in the interface of the second polypeptide isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity in the second polypeptide, wherein theprotuberance is positioned to interact with the cavity;

(c) introducing into a host cell the first and second nucleic acids andculturing the host cell so that expression of the first and secondnucleic acid occurs; and

(d) recovering the heteromultimeric antibody formed from the cellculture.

It may also be desirable to construct a multispecific antibody (such asa bispecific antibody) that incorporates a previously identifiedantibody. Under these circumstances it is desirable to identify a heavychain that when paired with the original light chain will bindspecifically to a second antigen of interest. The methods of Figini etal. (Figini, M. et al. (1994) J. Mol. Biol. 239:68-78, hereinincorporated by reference in its entirety) may be used to identify sucha heavy chain. First a phage library would be treated with guanidinehydrochloride to dissociate the original light chain. Next, the heavychains displayed on phage would be reconstituted with the light chain ofinterest by removing the denaturant (such as by dialysis). Panningagainst the second antigen of interest would then be conducted toidentify the desired heavy chain. The invention further embodies amultispecific antibody prepared by this method of selecting a heavychain to pair with a chosen light chain, nucleic acid encoding theantibody, and a host cell comprising the nucleic acid.

The invention provides a mechanism for increasing the yields of theheteromultimer over other unwanted end-products such as undesiredheteromultimers and/or homomultimers (see FIG. 1A-1C). Preferably, theyields of the desired heteromultimer recovered from recombinant cellculture are at least greater than 80% by weight and preferably greaterthan 90% by weight compared to the by-product undesired heterodimer orhomomultimer(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. FIG. 1A is a diagram of the formation of Fc-containingbispecific antibodies when no engineering is performed to enhanceheteromultimerization over homomultimerization. FIG. 1B is a diagramshowing pairing that occurs when heavy (H) chains are engineered suchthat desired heteromultimerization is favored over undesiredheteromultimerization over homomultimerization. FIG. 1C is a diagramshowing pairing that occurs when antibodies are chosen which share thesame light (L) chain to circumvent the problem of light chains pairingwith non-cognate heavy chains.

FIGS. 2A-2C. FIG. 2A diagrams a selection scheme for C_(H)3 heterodimerusing phage display vector, pRA2. Phage displaying stable C_(H)3heterodimers are captured using an antibody directed to the gD flag.FIG. 2B diagrams a dicistronic operon in which C_(H)3 expressed from asynthetic gene is co-secreted with a second copy of C_(H)3 expressedfrom the natural gene (Ellison et al. Nucleic Acids Res. 10:4071-4079(1982)) as a fusion protein with M13 gene III protein. The syntheticC_(H)3 gene is preceded by a sequence encoding a peptide derived fromherpes simplex virus glycoprotein D (gD flag, Lasky, L. A. and Dowbenko,D. J. (1984) DNA 3:23-29; Berman, P. W. et al., (1985) Science227:1490-1492 and a cleavage (G) site for the site-specific protease,Genenase I (Carter, P. et al. (1989) Proteins: Structure, Function andGenetics 6:240-248). FIG. 2C is the nucleic acid sequence of thedicistronic operon (SEQ ID NO:1) of FIG. 2B in which the residues in thetranslated C_(H)3 genes are numbered according to the Eu system of Kabatet al. In Sequences of Proteins of Immunological Interest, 5th ed. vol.1, pp. 688-696, NIH, Bethesda, Md. (1991). Protuberance mutation T366Wis shown, as are the residues targeted for randomization in the naturalC_(H)3 gene (366, 368, and 407).

FIGS. 3A-3C. FIGS. 3A and 3B are bar graphs of the results of scanningdensitometric analysis of SDS-PAGE of protein A-purified products fromcotransfection of antibody (Ab) heavy and light chains withimmunoadhesin (Ia). Data presented are the mean of two independentexperiments. The x-axis indicates the ratios of input DNA by mass(Ia:H:L) and the y-axis indicates the percentage of each type of productmultimer with respect to total product protein. FIG. 3C is a diagram ofthe possible product multimers.

FIG. 4 is a comparison of the V_(L) sequences of eight differentantibodies with specificities for Axl, Rse, IgER, Ob-R, and VEGF. Theposition of the antigen binding CDR residues according to sequencedefinition (Kabat et al. (1991) supra) or structural definition(Chothia, C. and Lesk, A. M. J. Mol. Biol. (1987) 196:901-917) are shownby underlining and #, respectively. Residues that differ from the Axl.78sequence are shown by double underlining.

FIG. 5 is a comparison of the heavy and light chains of selectedanti-Ob-R and anti-HER3 clones. Shown are the V_(H) and the common V_(L)sequences of anti-Ob-R clone 26 and anti-HER3 clone 18 used to constructa bispecific antibody.

FIG. 6. Sandwich ELISA for detection of simultaneous binding to Mpl-IgGand HER3-IgG. Antibodies tested were the anti-Mpl×anti-HER3 BsIgGcontaining the mutations, Y349C:T366S:L368A:Y407V/T366′W:S354° C.,together with corresponding parental anti-Mpl or anti-HER3 IgG withmutated Fc regions.

FIG. 7 is a bar graph of the results of an antibody-dependentcell-mediated cytotoxicity (ADCC) study. ADCC was mediated by huMAb4D5-5(Carter, P. et al. (1992) PNAS USA 89:4285-4289) containing either amutant (S354C:T366W/Y349′C:T366′S:L368′A:Y407′V) or wild-type Fc or anisotype-matched control antibody (E25, Presta, L. G. et al. (1993) J.Immunol. 151:2623-2632). The antibodies (125 ng/ml) were incubated withhuman peripheral blood mononuclear effector cells and SK-BR-3 targetcells at the ratios shown. Data presented are the mean of triplicatemeasurements and representative of three separate experiments.

FIG. 8 is a matrix representing the amino acid sequence identity betweenthe light chains of antibodies raised to HER3 versus the light chains ofantibodies raised to Ob-R. Antibodies having light chains with 100%sequence identity are indicated in blackened boxes. Antibodies havinglight chains with 98-99% sequence identity are indicated in white boxes.The antibody clone identity is indicated below the matrix.

I. Definitions

In general, the following words or phrases have the indicateddefinitions when used in the description, examples, and claims:

A “heteromultimer”, “heteromultimeric polypeptide”, or “heteromultimericmultispecific antibody” is a molecule comprising at least a firstpolypeptide and a second polypeptide, wherein the second polypeptidediffers in amino acid sequence from the first polypeptide by at leastone amino acid residue. Preferably, the heteromultimer has bindingspecificity for at least two different ligands or binding sites. Theheteromultimer can comprise a “heterodimer” formed by the first andsecond polypeptide or can form higher order tertiary structures wherepolypeptides in addition to the first and second polypeptide arepresent. Exemplary structures for the heteromultimer includeheterodimers (e.g. the bispecific immunoadhesin described by Dietsch etal., supra), heterotrimers (e.g. the Ab/Ia chimera described by Chamowet al., supra), heterotetramers (e.g. a bispecific antibody) and furtheroligomeric structures.

As used herein, “multimerization domain” refers to a region of each ofthe polypeptides of the heteromultimer. The “multimerization domain”promotes stable interaction of the chimeric molecules within theheteromultimer complex. Preferably, the multimerization domain promotesinteraction between a specific first polypeptide and a specific secondpolypeptide, thereby enhancing the formation of the desiredheteromultimer and substantially reducing the probability of theformation of undesired heteromultimers or homomultimers. Themultimerization domains may interact via an immunoglobulin sequence,leucine zipper, a hydrophobic region, a hydrophilic region, or a freethiol which forms an intermolecular disulfide bond between the chimericmolecules of the chimeric heteromultimer. The free thiol may beintroduced into the interface of one or more interacting polypeptides bysubstituting a naturally occurring residue of the polypeptide with, forexample, a cysteine at a position allowing for the formation of adisulfide bond between the polypeptides. The multimerization domain maycomprise an immunoglobulin constant region. A possible multimerizationdomain useful in the present invention is disclosed in PCT/US90/06849(herein incorporated by reference in its entirety) in which hybridimmunoglobulins are described. In addition a multimerization region maybe engineered such that steric interactions not only promote stableinteraction, but further promote the formation of heterodimers overhomodimers from a mixture of monomers. See, for example, PCT/US96/01598(herein incorporated by reference in its entirety) in which a“protuberance-into-cavity” strategy is disclosed for an interfacebetween a first and second polypeptide for hetero-oligomerization.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG₁, IgG₂, IgG₃or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferably IgG₁, IgG₂, IgG₃or IgG₄.

By “free thiol-containing compound” is meant a compound that can beincorporated into or reacted with an amino acid of a polypeptideinterface of the invention such that the free thiol moiety of thecompound is positioned to interact with a free thiol of moiety at theinterface of additional polypeptide of the invention to form a disulfidebond. Preferably, the free thiol-containing compound is cysteine.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising the entire chimeric heteroadhesin, or a fragmentthereof, fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with activity of thechimeric heteroadhesin. The tag polypeptide preferably is fairly uniqueso that the antibody thereagainst does not substantially cross-reactwith other epitopes. Suitable tag polypeptides generally have at least 6amino acid residues and usually between about 8-50 amino acid residues(preferably between about 9-30 residues). An embodiment of the inventionencompasses a chimeric heteroadhesin linked to an epitope tag, which tagis used to detect the adhesin in a sample or recover the adhesin from asample. As used herein, “common light chain” or “common amino acidsequence of the light chain” refers to the amino acid sequence of thelight chain in the multispecific antibody of the invention. Panels ofantibodies were generated against at least two different antigens bypanning a phage display library such as that described by Vaughan, etal. (1996) supra, herein incorporated by reference in its entirety withparticular reference to the method of selection of the phagemidlibrary). The light chain sequences were compared with respect to thevariable light chain amino acid sequences. Useful light chains from thecompared panels are those having amino acid sequence identity of atleast 80%, preferably at least 90%, more preferably at least 95%, andmost preferably 100% identity. A common light chain sequence is asequence designed to be an approximation of the two compared light chainsequences. Where the compared light chains are 100% sequence identicalat the amino acid level, the common light chain is identical to thelight chains from the selected library clones, even though the lightchain functions in a different binding domain of the multispecificantibody. Where the compared light chains differ as described above, thecommon light chain may differ from one or the other, or both, of thecompared light chains from the library clones. In a case in which thecommon light chain differs from one or the other, or both of the libraryclones, it is preferred that the differing residues occur outside of theantigen binding CDR residues of the antibody light chain. For example,the position of the antigen binding CDR residues may be determinedaccording to a sequence definition (Kabat et al. (1991) supra) orstructural definition (Chothia and Lesk (1987) J. Mol. Biol.196:901-917).

As used herein, “amino acid sequence identity” refers to the percentageof the amino acids of one sequence are the same as the amino acids of asecond amino acid sequence. 100% sequence identity between polypeptidechains means that the chains are identical.

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. Preferably, mammalianpolypeptides (polypeptides that were originally derived from a mammalianorganism) are used, more preferably those which are directly secretedinto the medium. Examples of bacterial polypeptides include, e.g.,alkaline phosphatase and β-lactamase. Examples of mammalian polypeptidesinclude molecules such as renin, a growth hormone, including humangrowth hormone; bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors;integrin; protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

The “first polypeptide” is any polypeptide which is to be associatedwith a second polypeptide. The first and second polypeptide meet at an“interface” (defined below). In addition to the interface, the firstpolypeptide may comprise one or more additional domains, such as“binding domains” (e.g. an antibody variable domain, receptor bindingdomain, ligand binding domain or enzymatic domain) or antibody constantdomains (or parts thereof) including C_(H)2, C_(H)1 and C_(L) domains.Normally, the first polypeptide will comprise at least one domain whichis derived from an antibody. This domain conveniently is a constantdomain, such as the C_(H)3 domain of an antibody and can form theinterface of the first polypeptide. Exemplary first polypeptides includeantibody heavy chain polypeptides, chimeras combining an antibodyconstant domain with a binding domain of a heterologous polypeptide(i.e. an immunoadhesin, see definition below), receptor polypeptides(especially those which form dimers with another receptor polypeptide,e.g., interleukin-8 receptor (IL-8R) and integrin heterodimers (e.g.LFA-1 or GPIIIb/IIIa)), ligand polypeptides (e.g. nerve growth factor(NGF), neurotrophin-3 (NT-3), and brain-derived neurotrophic factor(BDNF)—see Arakawa et al. J. Biol. Chem. 269(45): 27833-27839 (1994) andRadziejewski et al. Biochem. 32(48): 1350 (1993)) and antibody variabledomain polypeptides (e.g. diabodies). The preferred first polypeptide isselected from an antibody heavy chain fused to a constant domain of animmunoglobulin, wherein the constant domain has been altered at theinterface to promote preferential interaction with a second polypeptideof the invention.

The “second polypeptide” is any polypeptide which is to be associatedwith the first polypeptide via an “interface”. In addition to theinterface, the second polypeptide may comprise additional domains suchas a “binding domain” (e.g. an antibody variable domain, receptorbinding domain, ligand binding domain or enzymatic domain), or antibodyconstant domains (or parts thereof) including C_(H)2, C_(H)1 and C_(L)domains. Normally, the second polypeptide will comprise at least onedomain which is derived from an antibody. This domain conveniently is aconstant region, such as the C_(H)3 domain of an antibody and can formthe interface of the second polypeptide. Exemplary second polypeptidesinclude antibody heavy chain polypeptides, chimeras combining anantibody constant domain with a binding domain of a heterologouspolypeptide (i.e. an immunoadhesin, see definition below), receptorpolypeptides (especially those which form dimers with another receptorpolypeptide, e.g., interleukin-8 receptor (IL-8R) and integrinheterodimers (e.g. LFA-1 or GPIIIb/IIIa)), ligand polypeptides (e.g.nerve growth factor (NGF), neurotrophin-3 (NT-3), and brain-derivedneurotrophic factor (BDNF)— see Arakawa et al. J. Biol. Chem.269(45):27833-27839 (1994) and Radziejewski et al. Biochem. 32(48):1350(1993)) and antibody variable domain polypeptides (e.g. diabodies). Thepreferred second polypeptide is selected from an antibody heavy chainfused to a constant domain of an immunoglobulin, wherein the constantdomain has been altered at the interface to promote preferentialinteraction with a first polypeptide of the invention.

A “binding domain” comprises any region of a polypeptide which isresponsible for selectively binding to a molecule of interest (e.g. anantigen, ligand, receptor, substrate or inhibitor). Exemplary bindingdomains include an antibody variable domain, receptor binding domain,ligand binding domain and an enzymatic domain. In preferred embodiments,the binding domain includes an immunoglobulin heavy chain and lightchain. According to the bispecific antibodies of the invention and themethod of making them, the light chain for each binding domain of thebispecific antibody is a common light chain, thereby avoiding theformation of undesired hetermultimers in which mispairing of heavy andlight chains occurs.

The term “antibody” as it refers to the invention shall mean apolypeptide containing one or more domains that bind an epitope on anantigen of interest, where such domain(s) are derived from or havesequence identity with the variable region of an antibody. Examples ofantibodies include full length antibodies, antibody fragments, singlechain molecules, bispecific or bifunctional molecules, diabodies,chimeric antibodies (e.g. humanized and PRIMATIZED™ antibodies), andimmunoadhesins. “Antibody fragments” include Fv, Fv′, Fab, Fab′, andF(ab′)₂ fragments.

“Humanized” forms of non-human (e.g. rodent or primate) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, rabbit or primate having the desired specificity, affinityand capacity. In some instances, Fv framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, the humanized antibody may comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. These modifications are made to furtherrefine and maximize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody preferably also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. The humanized antibody includes a PRIMATIZED™antibody wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by immunizing macaque monkeys with the antigenof interest.

A “multispecific antibody” is a molecule having binding specificitiesfor at least two different antigens. While such molecules normally willonly bind two antigens (i.e. bispecific antibodies, BsAbs), antibodieswith additional specificities such as trispecific antibodies areencompassed by this expression when used herein. Examples of BsAbsinclude those with one arm directed against a tumor cell antigen and theother arm directed against a cytotoxic trigger molecule such asanti-FcγRI/anti-CD15, anti-p185^(HER2)/FcγRIII (CD16),anti-CD3/anti-malignant B-cell (1D10), anti-CD3/anti-p185^(HER2),anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma,anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which bindsspecifically to a tumor antigen and one arm which binds to a toxin suchas anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activatedprodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzesconversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbswhich can be used as fibrinolytic agents such as anti-fibrin/anti-tissueplasminogen activator (tPA), anti-fibrin/anti-urokinase-type plasminogenactivator (uPA); BsAbs for targeting immune complexes to cell surfacereceptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor(e.g. FcγRI, FcγRII or FcγRIII); BsAbs for use in therapy of infectiousdiseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cellreceptor: CD3 complex/anti-influenza, anti-FcγR/anti-HIV; BsAbs fortumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE,anti-CEA/anti-DPTA, anti-p185^(HER2)/anti-hapten; BsAbs as vaccineadjuvants (see Fanger et al., supra); and BsAbs as diagnostic tools suchas anti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase(HRP)/anti-hormone, anti-somatostatin/anti-substance P,anti-HRP/anti-FITC, anti-CEA/anti-β-galactosidase (see Nolan et al.,supra). Examples of trispecific antibodies includeanti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 andanti-CD3/anti-CD8/anti-CD37.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous protein(an “adhesin”, e.g. a receptor, ligand or enzyme) with the effectorfunctions of immunoglobulin constant domains. Structurally, theimmunoadhesins comprise a fusion of the adhesin amino acid sequence withthe desired binding specificity which is other than the antigenrecognition and binding site (antigen combining site) of an antibody(i.e. is “heterologous”) and an immunoglobulin constant domain sequence.The immunoglobulin constant domain sequence in the immunoadhesin may beobtained from any immunoglobulin, such as IgG₁, IgG₂, IgG₃, or IgG₄subtypes, IgA, IgE, IgD or IgM.

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding ability, and preferably thebiological activity of a corresponding native receptor. In a specificembodiment, the receptor is from a cell-surface polypeptide having anextracellular domain which is homologous to a member of theimmunoglobulin supergenefamily. Other typical receptors, are not membersof the immunoglobulin supergenefamily but are nonetheless specificallycovered by this definition, are receptors for cytokines, and inparticular receptors with tyrosine kinase activity (receptor tyrosinekinases), members of the hematopoietin and nerve growth factor receptorsuperfamilies, and cell adhesion molecules, e.g. (E-, L- and P-)selectins.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability, and preferably the biological activity of acorresponding native ligand. This definition, among others, specificallyincludes binding sequences from ligands for the above-mentionedreceptors.

As used herein the phrase “multispecific immunoadhesin” designatesimmunoadhesins (as hereinabove defined) having at least two bindingspecificities (i.e. combining two or more adhesin binding domains).Multispecific immunoadhesins can be assembled as heterodimers,heterotrimers or heterotetramers, essentially as disclosed in WO89/02922 (published 6 Apr. 1989), in EP 314,317 (published 3 May 1989),and in U.S. Pat. No. 5,116,964 issued 2 May 1992. Preferredmultispecific immunoadhesins are bispecific. Examples of bispecificimmunoadhesins include CD4-IgG/TNFreceptor-IgG andCD4-IgG/L-selectin-IgG. The last mentioned molecule combines the lymphnode binding function of the lymphocyte homing receptor (LHR,L-selectin), and the HIV binding function of CD4, and finds potentialapplication in the prevention or treatment of HIV infection, relatedconditions, or as a diagnostic.

An “antibody-immunoadhesin chimera (Ab/Ia chimera)” comprises a moleculewhich combines at least one binding domain of an antibody (as hereindefined) with at least one immunoadhesin (as defined in thisapplication). Exemplary Ab/Ia chimeras are the bispecific CD4-IgGchimeras described by Berg et al., supra and Chamow et al., supra.

The “interface” comprises those “contact” amino acid residues (or othernon-amino acid groups such as carbohydrate groups, NADH, biotin, FAD orhaem group) in the first polypeptide which interact with one or more“contact” amino acid residues (or other non-amino acid groups) in theinterface of the second polypeptide. The preferred interface is a domainof an immunoglobulin such as a variable domain or constant domain (orregions thereof), however the interface between the polypeptides forminga heteromultimeric receptor or the interface between two or more ligandssuch as NGF, NT-3 and BDNF are included within the scope of this term.The preferred interface comprises the C_(H)3 domain of an immunoglobulinwhich preferably is derived from an IgG antibody and most preferably ahuman IgG₁ antibody.

An “original” amino acid residue is one which is replaced by an “import”residue which can have a smaller or larger side chain volume than theoriginal residue. The import amino acid residue can be a naturallyoccurring or non-naturally occurring amino acid residue, but preferablyis the former. “Naturally occurring” amino acid residues are thoseresidues encoded by the genetic code and listed in Table 1 ofPCT/US96/01598, herein incorporated by reference in its entirety. By“non-naturally occurring” amino acid residue is meant a residue which isnot encoded by the genetic code, but which is able to covalently bindadjacent amino acid residue(s) in the polypeptide chain. Examples ofnon-naturally occurring amino acid residues are norleucine, ornithine,norvaline, homoserine and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym. 202:301-336 (1991), forexample. To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244: 182 (1989) and Ellman etal., supra can be used. Briefly, this involves chemically activating asuppressor tRNA with a non-naturally occurring amino acid residuefollowed by in vitro transcription and translation of the RNA. Themethod of the instant invention involves replacing at least one originalamino acid residue, but more than one original residue can be replaced.Normally, no more than the total residues in the interface of the firstor second polypeptide will comprise original amino acid residues whichare replaced. The preferred original residues for replacement are“buried”. By “buried” is meant that the residue is essentiallyinaccessible to solvent. The preferred import residue is not cysteine toprevent possible oxidation or mispairing of disulfide bonds. By“original nucleic acid” is meant the nucleic acid encoding a polypeptideof interest which can be altered to encode within the multimerizationdomain amino acids whose side chains interact at the interface betweenthe first and second polypeptide promoting stable interaction betweenthe polypeptides. Such alterations may generate without limitation suchstable interactions as protuberance-into-cavity, non-naturally occurringdisulfide bonds, leucine zipper, hydrophobic interactions, andhydrophilic interations. Preferably, the alteration is chosen whichpromotes specific interaction between a first and second polypeptide ofinterest and effectively excludes interactions that result in undesiredheteromer pairing or the formation of homomers. The original or startingnucleic acid may be a naturally occurring nucleic acid or may comprise anucleic acid which has been subjected to prior alteration (e.g. ahumanized antibody fragment). By “altering” the nucleic acid is meantthat the original nucleic acid is genetically engineered or mutated byinserting, deleting or replacing at least one codon encoding an aminoacid residue of interest. Normally, a codon encoding an original residueis replaced by a codon encoding an import residue. Techniques forgenetically modifying a DNA in this manner have been reviewed inMutagenesis: a Practical Approach, M.J. McPherson, Ed., (IRL Press,Oxford, UK. (1991), and include site-directed mutagenesis, cassettemutagenesis and polymerase chain reaction (PCR) mutagenesis, forexample.

The protuberance, cavity, or free thiol (such as a cysteine residue fordisulfide bond formation) can be “introduced” into the interface of thefirst or second polypeptide by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. According, the protuberance, cavity or free thiol which is“introduced” is “non-naturally occurring” or “non-native”, which meansthat it does not exist in nature or in the original polypeptide (e.g. ahumanized monoclonal antibody). Preferably the import amino acid residuefor forming the protuberance has a relatively small number of “rotamers”(e.g. about 3-6). A “rotamer” is an energetically favorable conformationof an amino acid side chain. The number of rotamers of the various aminoacid residues are reviewed in Ponders and Richards, J. Mol. Biol.193:775-791 (1987).

“Isolated” heteromultimer means heteromultimer which has been identifiedand separated and/or recovered from a component of its natural cellculture environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the heteromultimer, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, theheteromultimer will be purified (1) to greater than 95% by weight ofprotein as determined by the Lowry method, and most preferably more than99% by weight, (2) to a degree sufficient to obtain at least 15 residuesof N-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. The heteromultimers of the present invention are generallypurified to substantial homogeneity. The phrases “substantiallyhomogeneous”, “substantially homogeneous form” and “substantialhomogeneity” are used to indicate that the product is substantiallydevoid of by-products originated from undesired polypeptide combinations(e.g. homomultimers). Expressed in terms of purity, substantialhomogeneity means that the amount of by-products does not exceed 10%,and preferably is below 5%, more preferably below 1%, most preferablybelow 0.5%, wherein the percentages are by weight.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

II. Preparation of the Heteromultimer

1. Preparation of the Starting Materials

As a first step, the first and second polypeptide (and any additionalpolypeptides forming the heteromultimer) are selected. Normally, thenucleic acid encoding these polypeptides needs to be isolated so that itcan be altered to encode the protuberance or cavity, or both, as hereindefined. However, the mutations can be introduced using synthetic means,e.g. by using a peptide synthesizer. Also, in the case where the importresidue is a non-naturally occurring residue, the method of Noren etal., supra is available for making polypeptides having suchsubstitutions. Additionally, part of the heteromultimer is suitably maderecombinantly in cell culture and other part(s) of the molecule are madeby those techniques mentioned above. Techniques for isolating antibodiesand preparing immunoadhesins follow. However, it will be appreciatedthat the heteromultimer can be formed from, or incorporate, otherpolypeptides using techniques which are known in the art. For example,nucleic acid encoding a polypeptide of interest (e.g. a ligand, receptoror enzyme) can be isolated from a cDNA library prepared from tissuebelieved to possess the polypeptide mRNA and to express it at adetectable level. Libraries are screened with probes (such as antibodiesor oligonucleotides of about 20-80 bases) designed to identify the geneof interest or the protein encoded by it. Screening the cDNA or genomiclibrary with the selected probe may be conducted using standardprocedures as described in chapters 10-12 of Sambrook et al., MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989).

(I) Antibody Preparation

Several techniques for the production of antibodies have been describedwhich include the traditional hybridoma method for making monoclonalantibodies, recombinant techniques for making antibodies (includingchimeric antibodies, e.g. humanized antibodies), antibody production intransgenic animals and the recently described phage display technologyfor preparing “fully human” antibodies. These techniques shall bedescribed briefly below. Polyclonal antibodies to the antigen ofinterest generally can be raised in animals by multiple subcutaneous(sC) or intraperitoneal (ip) injections of the antigen and an adjuvant.It may be useful to conjugate the antigen (or a fragment containing thetarget amino acid sequence) to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups. Animals areimmunized against the immunogenic conjugates or derivatives by combining1 mg of 1 μg of conjugate (for rabbits or mice, respectively) with 3volumes of Freud's complete adjuvant and injecting the solutionintradermally at multiple sites. One month later the animals are boostedwith ⅕ to 1/10 the original amount of conjugate in Freud's completeadjuvant by subcutaneous injection at multiple sites. 7 to 14 days laterthe animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are used to enhance theimmune response.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies using the hybridoma method first described byKohler and Milstein, Nature 256:495 (1975) or may be made by recombinantDNA methods (Cabilly et al., U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as hamster, isimmunized as hereinabove described to elicit lymphocytes that produce,or are capable of producing, antibodies that will specifically bind tothe protein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus preparedare seeded and grown in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, parental myeloma cells. For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (HATmedium), which substances prevent the growth of HGPRT-deficient cells.Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63, MarcelDekker, Inc., New York, 1987). See, also, Boerner et al., J. Immunol.,147(1):86-95 (1991) and WO 91/17769, published Nov. 28, 1991, fortechniques for the production of human monoclonal antibodies. Culturemedium in which hybridoma cells are growing is assayed for production ofmonoclonal antibodies directed against the antigen of interest.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson and Pollard, Anal. Biochem. 107:220 (1980). After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity, and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods. Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-104 (Academic Press, 1986).Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium or RPMI-1640 medium. In addition, the hybridomacells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography. Alternatively, it is now possibleto produce transgenic animals (e.g. mice) that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy chainjoining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA 90:2551-255 (1993); Jakobovits et al., Nature 362:255-258(1993); Fishwild, D. M., et al. (1996) Nat. Biotech 14:845-851; andMendez, M. J., et al. (1997) Nat. Genetics 15:146-156).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), using theantigen of interest to select for a suitable antibody or antibodyfragment. Clackson et al., Nature, 352:624-628 (1991) and Marks et al.,J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Mark et al., Bio/Technol. 10:779-783(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nuc. Acids Res., 21:2265-2266 (1993); Griffiths, A. D., et al.(1994) EMBO J. 13:3245-3260; and Vaughan, et al. (1996) supra). Thus,these techniques are viable alternatives to traditional monoclonalantibody hybridoma techniques for isolation of “monoclonal” antibodies(especially human antibodies) which are encompassed by the presentinvention.

DNA encoding the antibodies of the invention is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences, Morrisonet al., Proc. Nat. Acad. Sci. 81:6851 (1984). In that manner, “chimeric”antibodies are prepared that have the binding specificity of ananti-antigen monoclonal antibody herein.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino is acid residuesintroduced into it from a source which is non-human. Humanization can beperformed essentially following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues,and possibly some FR residues, are substituted by residues fromanalogous sites in rodent antibodies. It is important that antibodies behumanized with retention of high affinity for the antigen and otherfavorable biological properties. To achieve this goal, according to apreferred method, humanized antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three dimensional models of the parental and humanizedsequences. Three dimensional immunoglobulin models are familiar to thoseskilled in the art. Computer programs are available which illustrate anddisplay probable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. For further details see WO 92/22653, published Dec. 23, 1992.

(ii) Immunoadhesin Preparation

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982);EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Köhler etal., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res.41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984);Morrison, Science 229:1202 (1985); Morrison et al., Proc. Natl. Acad.Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559.Reassorted immunoglobulin chains also are known. See, for example, U.S.Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references citedtherein.

Chimeras constructed from an adhesin binding domain sequence linked toan appropriate immunoglobulin constant domain sequence (immunoadhesins)are known in the art. Immunoadhesins reported in the literature includefusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci.USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the hinge and Fc regions of an immunoglobulin heavychain. Ordinarily, when preparing the immunoadhesins of the presentinvention, nucleic acid encoding the binding domain of the adhesin willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the Ia.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG₁, IgG₂, orIgG₃ heavy chain. The precise site at which the fusion is made is notcritical, and the optimal site can be determined by routineexperimentation.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L)-AC_(L);

(b) AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H)];

(c) AC_(L)-AC_(H)-[AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H),or V_(L)C_(L)-V_(H)C_(H)];

(d) AC_(L)-V_(H)C_(H)-[AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H)];

(e) V_(L)C_(L)-AC_(H)-[AC_(L)-V_(H)C_(H) (or V_(L)C_(L)-AC_(H)]; and

(f) [A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

An immunoglobulin light chain might be present either covalentlyassociated to an adhesin-immunoglobulin heavy chain fusion polypeptide,or directly fused to the adhesin. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the adhesin-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodssuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567, issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG₁ and IgG₃ immunoglobulin sequencesis preferred. A major advantage of using IgG₁ is that IgG₁immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG₃ requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG₃ hingeis longer and more flexible, so it can accommodate larger “adhesin”domains that may not fold or function properly when fused to IgG₁.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG₁, IgG₂ and IgG₄ all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG₄ does not activate complement,and IgG₂ is significantly weaker at complement activation than IgG₁.Moreover, unlike IgG₁, IgG₂ does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG₃ is optimal for complement activation,its in vivo half-life is approximately one third of the other IgGisotypes. Another important consideration for immunoadhesins designed tobe used as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG₁ hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites, G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3 m5, 11 and 21) have one allotype which isnonimmunogenic. Thus, the potential immunogenicity of a γ3 immunoadhesinis greater than that of a γ1 immunoadhesin.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an Ig cDNA sequence.However, fusion to genomic Ig fragments can also be used (see, e.g.Gascoigne et al., supra; Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and the Igparts of the immunoadhesin are inserted in tandem into a plasmid vectorthat directs efficient expression in the chosen host cells.

2. Generating a Protuberance and/or Cavity

As a first step to selecting original residues for forming theprotuberance and/or cavity, the three-dimensional structure of theheteromultimer is obtained using techniques which are well known in theart such as X-ray crystallography or NMR. Based on the three-dimensionalstructure, those skilled in the art will be able to identify theinterface residues. The preferred interface is the C_(H)3 domain of animmunoglobulin constant domain. The interface residues of the C_(H)3domains of IgG, IgA, IgD, IgE and IgM have been identified (see, forexample, PCT/US96/01598, herein incorporated by reference in itsentirety), including those which are optimal for replacing with importresidues; as were the interface residues of various IgG subtypes and“buried” residues. The basis for engineering the C_(H)3 interface isthat X-ray crystallography has demonstrated that the intermolecularassociation between human IgG₁ heavy chains in the Fc region includesextensive protein/protein interaction between C_(H)3 domains whereas theglycosylated C_(H)2 domains interact via their carbohydrate(Deisenhofer, Biochem. 20:2361-2370 (1981)). In addition there are twointer-heavy chain disulfide bonds which are efficiently formed duringantibody expression in mammalian cells unless the heavy chain istruncated to remove C_(H)2 and C_(H)3 domains (King et al., Biochem. J.281:317 (1992)). Thus, heavy chain assembly appears to promote disulfidebond formation rather than vice versa. Taken together these structuraland functional data led to the hypothesis that antibody heavy chainassociation is directed by the C_(H)3 domains. It was further speculatedthat the interface between C_(H)3 domains might be engineered to promoteformation of heteromultimers of different heavy chains and hinderassembly of corresponding homomultimers. The experiments describedherein demonstrated that it was possible to promote the formation ofheteromultimers over homomultimers using this approach. Thus, it ispossible to generate a polypeptide fusion comprising a polypeptide ofinterest and the C_(H)3 domain of an antibody to form a first or secondpolypeptide. The preferred C_(H)3 domain is derived from an IgGantibody, such as an human IgG₁. Those interface residues which canpotentially constitute candidates for forming the protuberance or cavityare identified. It is preferable to select “buried” residues to bereplaced. To determine whether a residue is buried, the surfaceaccessibility program of Lee et al. J. Mol. Biol. 55:379-400 (1971) canbe used to calculate the solvent accessibility (SA) of residues in theinterface. Then, the SA for the residues of each of the first and secondpolypeptide can be separately calculated after removal of the otherpolypeptide. The difference in SA of each residue between the monomerand dimer forms of the interface can then be calculated using theequation: SA (dimer)-SA (monomer). This provides a list of residueswhich lose SA on formation of the dimer. The SA of each residue in thedimer is compared to the theoretical SA of the same amino acid in thetripeptide Gly-X-Gly, where X=the amino acid of interest (Rose et al.Science 229:834-838 (1985)). Residues which (a) lost SA in the dimercompared to the monomer and (b) had an SA less than 26% of that in theircorresponding tripeptide are considered as interface residues. Twocategories may be delineated: those which have an SA<10% compared totheir corresponding tripeptide (i.e. “buried”) and those which have25%>SA>10% compared to their corresponding tripeptide (i.e. “partiallyburied”) (see Table 1, below).

TABLE 1 SA Lost Monomer → Dimer % Tripeptide Residue PolypeptidePolypeptide Polypeptide Polypeptide No.^(†) A B A B Q347 22.1 31.0 25.026.5 Y349 79.8 83.9 5.2 5.7 L351 67.4 77.7 3.9 2.0 S354 53.4 52.8 11.311.7 E357 43.7 45.3 0.4 1.3 S364 21.5 15.1 0.5 1.4 T366 29.3 25.8 0.00.1 L368 25.5 29.7 1.0 1.1 K370 55.8 62.3 11.5 11.0 T394 64.0 58.5 0.61.4 V397 50.3 49.5 13.2 11.0 D399 39.7 33.7 5.7 5.7 F405 53.7 52.1 0.00.0 Y407 89.1 90.3 0.0 0.0 K409 86.8 92.3 0.7 0.6 T411 4.3 7.5 12.7 9.8^(†)residue numbering as in IgG crystal structure (Deisenhofer,Biochemistry 20: 2361-2370 (1981)).

The effect of replacing residues on the polypeptide chain structure canbe studied using a molecular graphics modeling program such as theInsight™ program (Biosym Technologies). Using the program, those buriedresidues in the interface of the first polypeptide which have a smallside chain volume can be changed to residues having a larger side chainvolume (i.e. a protuberance), for example. Then, the residues in theinterface of the second polypeptide which are in proximity to theprotuberance are examined to find a suitable residue for forming thecavity. Normally, this residue will have a large side chain volume andis replaced with a residue having a smaller side chain volume. Incertain embodiments, examination of the three-dimensional structure ofthe interface will reveal a suitably positioned and dimensionedprotuberance on the interface of the first polypeptide or a cavity onthe interface of the second polypeptide. In these instances, it is onlynecessary to model a single mutant, i.e., with a syntheticallyintroduced protuberance or cavity.

With respect to selecting potential original residues for replacementwhere the first and second polypeptide each comprise a C_(H)3 domain,the C_(H)3/C_(H)3 interface of human IgG₁ involves sixteen residues oneach domain located on four anti-parallel β-strands which buries 1090 Å²from each surface (Deisenhofer, supra) and Miller, J. Mol. Biol. 216:965(1990)). Mutations are preferably targeted to residues located on thetwo central anti-parallel β-strands. The aim is to minimize the riskthat the protuberances which are created can be accommodated byprotruding into surrounding solvent rather than by compensatory cavitiesin the partner C_(H)3 domain.

Once the preferred original/import residues are identified by molecularmodeling, the amino acid replacements are introduced into thepolypeptide using techniques which are well known in the art. Normallythe DNA encoding the polypeptide is genetically engineered using thetechniques described in Mutagenesis: a Practical Approach, supra.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution variants of the DNA encoding the first or secondpolypeptide. This technique is well known in the art as described byAdelman et al., DNA, 2:183 (1983). Briefly, first or second polypeptideDNA is altered by hybridizing an oligonucleotide encoding the desiredmutation to a DNA template, where the template is the single-strandedform of a plasmid or bacteriophage containing the unaltered or nativeDNA sequence of heteromultimer. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat will thus incorporate the oligonucleotide primer, and will code forthe selected alteration in the heteromultimer DNA.

Cassette mutagenesis can be performed as described Wells et al. Gene34:315 (1985) by replacing a region of the DNA of interest with asynthetic mutant fragment generated by annealing complimentaryoligonucleotides. PCR mutagenesis is also suitable for making variantsof the first or second polypeptide DNA. While the following discussionrefers to DNA, it is understood that the technique also findsapplication with RNA. The PCR technique generally refers to thefollowing procedure (see Erlich, Science, 252:1643-1650 (1991), thechapter by R. Higuchi, p. 61-70).

This invention also encompasses, in addition to the protuberance orcavity mutations, amino acid sequence variants of the heteromultimerwhich can be prepared by introducing appropriate nucleotide changes intothe heteromultimer DNA, or by synthesis of the desired heteromultimerpolypeptide. Such variants include, for example, deletions from, orinsertions or substitutions of, residues within the amino acid sequencesof the first and second polypeptides forming the heteromultimer. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired antigen-binding characteristics. The amino acid changes also mayalter post-translational processes of the heteromultimer, such aschanging the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of theheteromultimer polypeptides that are preferred locations for mutagenesisis called “alanine scanning mutagenesis,” as described by Cunningham andWells, Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g. charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the surrounding aqueous environment in or outsidethe cell. Those domains demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined.

Normally the mutations will involve conservative amino acid replacementsin non-functional regions of the heteromultimer. Exemplary mutations areshown in Table 2.

TABLE 2 Original Residue Exemplary Substitutions Preferred SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) AspAsp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu;Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val; IleMet; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T)Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile;Leu; Met; Phe; Ala; Leu Norleucine

Covalent modifications of the heteromultimer polypeptides are includedwithin the scope of this invention. Covalent modifications of theheteromultimer can be introduced into the molecule by reacting targetedamino acid residues of the heteromultimer or fragments thereof with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues. Another type of covalentmodification of the heteromultimer polypeptide included within the scopeof this invention comprises altering the native glycosylation pattern ofthe polypeptide. By altering is meant deleting one or more carbohydratemoieties found in the original heteromultimer, and/or adding one or moreglycosylation sites that are not present in the original heteromultimer.Addition of glycosylation sites to the heteromultimer polypeptide isconveniently accomplished by altering the amino acid sequence such thatit contains one or more N-linked glycosylation sites. The alteration mayalso be made by the addition of, or substitution by, one or more serineor threonine residues to the original heteromultimer sequence (forO-linked glycosylation sites). For ease, the heteromultimer amino acidsequence is preferably altered through changes at the DNA level,particularly by mutating the DNA encoding the heteromultimer polypeptideat preselected bases such that codons are generated that will translateinto the desired amino acids. Another means of increasing the number ofcarbohydrate moieties on the heteromultimer polypeptide is by chemicalor enzymatic coupling of glycosides to the polypeptide. These methodsare described in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal ofcarbohydrate moieties present on the heteromultimer may be accomplishedchemically or enzymatically.

Another type of covalent modification of heteromultimer compriseslinking the heteromultimer polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Since it is often difficult to predict in advance the characteristics ofa variant heteromultimer, it will be appreciated that some screening ofthe recovered variant will be needed to select the optimal variant.

3. Expression of Heteromultimer Having Common Light Chains

Following mutation of the DNA and selection of the common light chain asdisclosed herein, the DNA encoding the molecules is expressed usingrecombinant techniques which are widely available in the art. Often, theexpression system of choice will involve a mammalian cell expressionvector and host so that the heteromultimer is appropriately glycosylated(e.g. in the case of heteromultimers comprising antibody domains whichare glycosylated). However, the molecules can also be produced in theprokaryotic expression systems elaborated below. Normally, the host cellwill be transformed with DNA encoding both the first polypeptide, thesecond polypeptide, the common light chain polypeptide, and otherpolypeptide(s) required to form the heteromultimer, on a single vectoror independent vectors. However, it is possible to express the firstpolypeptide, second polypeptide, and common light chain polypeptide (theheteromultimer components) in independent expression systems and couplethe expressed polypeptides in vitro.

The nucleic acid(s) (e.g., cDNA or genomic DNA) encoding theheteromultimer and common light chain is inserted into a replicablevector for further cloning (amplification of the DNA) or for expression.Many vectors are available. The vector components generally include, butare not limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The polypeptides of the heteromultimer components may be produced asfusion polypeptides with a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the DNA that is inserted into the vector.The heterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells, the signal sequence may besubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the is alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,alpha factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182 issued 23 Apr.1991), or acid phosphatase leader, the C. albicans glucoamylase leader(EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression the nativesignal sequence (e.g., the antibody or adhesin presequence that normallydirects secretion of these molecules from human cells in vivo) issatisfactory, although other mammalian signal sequences may be suitableas well as viral secretory leaders, for example, the herpes simplex gDsignal. The DNA for such precursor region is ligated in reading frame toDNA encoding the polypeptides forming the heteromultimer.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet. 1:327(1982)), mycophenolic acid (Mulligan et al., Science 209:1422 (1980)) orhygromycin (Sugden et al., Mol. Cell. Biol. 5:410-413 (1985)). The threeexamples given above employ bacterial genes under eukaryotic control toconvey resistance to the appropriate drug G418 or neomycin (geneticin),xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theheteromultimer nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformants are placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodesheteromultimer. Increased quantities of heteromultimer are synthesizedfrom the amplified DNA. Other examples of amplifiable genes includemetallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216 (1980). The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding thecomponents of the heteromultimer. This amplification technique can beused with any otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1,notwithstanding the presence of endogenous DHFR if, for example, amutant DHFR gene that is highly resistant to Mtx is employed (EP117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding heteromultimer, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979);Kingsman et al., Gene 7:141 (1979); or Tschemper et al., Gene 10:157(1980)). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,Curr. Genet. 12:185 (1987). More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, Bio/Technology 8:135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed(Fleer et al., Bio/Technology 9:968-975 (1991)).

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theheteromultimer nucleic acid. A large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to heteromultimer-encoding DNA by removing the promoterfrom the source DNA by restriction enzyme digestion and inserting theisolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 (1980) and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the heteromultimer (Siebenlistet al., Cell 20:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the heteromultimer.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968); and Holland, Biochemistry 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Heteromultimer transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter orfrom heat-shock promoters.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature 273:113 (1978); Mulligan and Berg,Science 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA 78:7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene 18:355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

Transcription of DNA encoding the heteromultimer components by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are relatively orientation and position independent,having been found 5′ (Laimins et al., Proc. Natl. Acad. Sci. USA 78:993(1981)) and 3′ (Lusky et al., Mol. Cell. Bio. 3:1108 (1983)) to thetranscription unit, within an intron (Banerji et al., Cell 33:729(1983)), as well as within the coding sequence itself (Osborne et al.,Mol. Cell. Bio. 4:1293 (1984)). Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theheteromultimer-encoding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the heteromultimer.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology 65:499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding heteromultimer. In general, transient expression involvesthe use of an expression vector that is able to replicate efficiently ina host cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Sambrook et al., supra,pp. 16.17-16.22. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of heteromultimers having desired bindingspecificities/affinities or the desired gel migration characteristicsrelative to heteromultimers or homomultimers lacking the non-naturaldisulfide bonds generated according to the instant invention.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the heteromultimer in recombinant vertebrate cell cultureare described in Gething et al., Nature 293:620-625 (1981); Mantei etal., Nature 281:40-46 (1979); EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of theheteromultimer is pRK5 (EP 307,247) or pSVI6B (PCT pub. no. WO 91/08291published 13 Jun. 1991).

The choice of host cell line for the expression of heteromultimerdepends mainly on the expression vector. Another consideration is theamount of protein that is required. Milligram quantities often can beproduced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5-based vectors by a modification of the calciumphosphate method to allow efficient heteromultimer expression.CDM8-based vectors can be used to transfect COS cells by theDEAE-dextran method (Aruffo et al., Cell 61:1303-1313 (1990); andZettmeissl et al., DNA Cell Biol. (US) 9:347-353 (1990)). If largeramounts of protein are desired, the immunoadhesin can be expressed afterstable transfection of a host cell line. For example, a pRK5-basedvector can be introduced into Chinese hamster ovary (CHO) cells in thepresence of an additional plasmid encoding dihydrofolate reductase(DHFR) and conferring resistance to G418. Clones resistant to G418 canbe selected in culture. These clones are grown in the presence ofincreasing levels of DHFR inhibitor methotrexate and clones are selectedin which the number of gene copies encoding the DHFR and heteromultimersequences is co-amplified. If the immunoadhesin contains a hydrophobicleader sequence at its N-terminus, it is likely to be processed andsecreted by the transfected cells. The expression of immunoadhesins withmore complex structures may require uniquely suited host cells. Forexample, components such as light chain or J chain may be provided bycertain myeloma or hybridoma host cells (Gascoigne et al., supra; andMartin et al., J. Virol. 67:3561-3568 (1.993)).

Other suitable host cells for cloning or expressing the vectors hereinare prokaryote, yeast, or other higher eukaryote cells described above.Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains.such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 is aparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 27C7. The complete genotype of 27C7 is tonAΔ ptr3 phoAΔE15Δ(argF-lac)169 ompTΔ degP41kan^(r). Strain 27C7 was deposited on 30 Oct.1991 in the American Type Culture Collection as ATCC No. 55,244.Alternatively, the strain of E. coli having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 may be employed.Alternatively, methods of cloning, e.g., PCR or other nucleic acidpolymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forheteromultimer-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach and Nurse, Nature 290:140 (1981); EP 139,383 published May2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,supra) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourtet al., J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., supra), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:265-278 (1988));Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case etal., Proc. Natl. Acad. Sci. USA 76:5259-5263 (1979)); Schwanniomycessuch as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillushosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.Commun. 112:284-289 (1983); Tilburn et al., Gene 26:205-221 (1983);Yelton et al., Proc. Natl. Acad. Sci. USA 81:1470-1474 (1984)) and A.niger (Kelly and Hynes, EMBO J. 4:475-479 (1985)).

Suitable host cells for the expression of glycosylated heteromultimerare derived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature315:592-594 (1985). A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the heteromultimer DNA. During incubation of the plant cellculture with A. tumefaciens, the DNA encoding the heteromultimer istransferred to the plant cell host such that it is transfected, andwill, under appropriate conditions, express the heteromultimer DNA. Inaddition, regulatory and signal sequences compatible with plant cellsare available, such as the nopaline synthase promoter andpolyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen.1:561 (1982). In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. EP 321,196 published 21 Jun. 1989.

The preferred hosts are vertebrate cells, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure inrecent years (Tissue Culture, Academic Press, Kruse and Patterson,editors (1973)). Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected with the above-described expression orcloning vectors of this invention and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.Depending on the host cell used, transfection is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described in section 1.82 of Sambrook et al.,supra, or electroporation is generally used for prokaryotes or othercells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene 23:315 (1983) and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued 16 Aug. 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact. 130:946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76:3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, or polycations, e.g., polybrene, polyornithine, etc.,may also be used. For various techniques for transforming mammaliancells, see Keown et al., Methods in Enzymology (1989), Keown et al.,Methods in Enzymology 185:527-537 (1990), and Mansour et al., Nature336:348-352 (1988).

Prokaryotic cells used to produce the heteromultimer polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the heteromultimer of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz. 58:44 (1979),Barnes and Sato, Anal. Biochem. 102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of allof which are incorporated herein by reference, may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed., IRLPress, 1991.

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

4. Recovery of the Heteromultimer

The heteromultimer preferably is generally recovered from the culturemedium as a secreted polypeptide, although it also may be recovered fromhost cell lysate when directly produced without a secretory signal. Ifthe heteromultimer is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100).

When the heteromultimer is produced in a recombinant cell other than oneof human origin, it is completely free of proteins or polypeptides ofhuman origin. However, it is necessary to purify the heteromultimer fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to heteromultimer. As a first step, theculture medium or lysate is normally centrifuged to remove particulatecell debris.

Heterodimers having antibody constant domains can be convenientlypurified by hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography, with affinity chromatography beingthe preferred purification technique. Where the heteromultimer comprisesa C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg,N.J.) is useful for purification. Other techniques for proteinpurification such as fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin Sepharose, chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the polypeptide to be recovered. The suitabilityof protein A as an affinity ligand depends on the species and isotype ofthe immunoglobulin Fc domain that is used in the chimera. Protein A canbe used to purify immunoadhesins that are based on human γ1, γ2, or γ4heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).Protein G is recommended for all mouse isotypes and for human γ3 (Gusset al., EMBO J. 5:15671575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. The conditions forbinding an immunoadhesin to the protein A or G affinity column aredictated entirely by the characteristics of the Fc domain; that is, itsspecies and isotype. Generally, when the proper ligand is chosen,efficient binding occurs directly from unconditioned culture fluid. Onedistinguishing feature of immunoadhesins is that, for human γ1molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in a heterodimer preparation that is >95%pure.

5. Uses for a Heteromultimeric Multispecific Antibody Having CommonLight Chains

Many therapeutic applications for the heteromultimer are contemplated.For example, the heteromultimer can be used for redirected cytotoxicity(e.g. to kill tumor cells), as a vaccine adjuvant, for deliveringthrombolytic agents to clots, for converting enzyme activated prodrugsat a target site (e.g. a tumor), for treating infectious diseases,targeting immune complexes to cell surface receptors, or for deliveringimmunotoxins to tumor cells. For example, tumor vasculature targetinghas been accomplished by targeting a model endothelial antigen, class IImajor histocompatibility complex, with an antibody-ricin immunotoxin(Burrows, F.J. and Thorpe, P.E. (1993) Proc Natl Acad Sci USA90:8996-9000). Significantly greater efficacy was achieved by combiningthe anti-endothelial immunotoxin with a second immunotoxin directedagainst the tumor cells themselves (Burrows, F. J. and Thorpe, P. E.(1993) supra). Recently, tissue factor was successfully targeted totumor vasculature using a bispecific antibody, triggering localthrombosis that resulted in significant anti-tumor efficacy (Huang, X.et al. (1997) Science 275:547-550). In addition, bispecific diabodieshave been used successfully to direct cytotoxic T-cells to kill targetbreast tumor cells and B-cell lymphoma cells in vitro (Zhu, Z. et al.(1996) Bio/Technology 14:192-196; and Holliger, P. et al. (1996) ProteinEngin. 9:299-305).

Therapeutic formulations of the heteromultimer are prepared for storageby mixing the heteromultimer having the desired degree of purity withoptional physiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The heteromultimer also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The heteromultimer to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The heteromultimer ordinarily will be stored inlyophilized form or in solution.

Therapeutic heteromultimer compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of heteromultimer administration is in accord with knownmethods, e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems as noted below.The heteromultimer is administered continuously by infusion or by bolusinjection.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) andLanger, Chem. Tech. 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release heteromultimer compositions also include liposomallyentrapped heteromultimer. Liposomes containing heteromultimer areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal heteromultimer therapy.

An effective amount of heteromultimer to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.A typical daily dosage might range from about 1 μg/kg to up to 10 mg/kgor more, depending on the factors mentioned above. Typically, theclinician will administer heteromultimer until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays.

The heteromultimers described herein can also be used in enzymeimmunoassays. To achieve this, one arm of the heteromultimer can bedesigned to bind to a specific epitope on the enzyme so that bindingdoes not cause enzyme inhibition, the other arm of the heteromultimercan be designed to bind to the immobilizing matrix ensuring a highenzyme density at the desired site. Examples of such diagnosticheteromultimers include those having specificity for IgG as well asferritin, and those having binding specificities for horse radishperoxidase (HRP) as well as a hormone, for example.

The heteromultimers can be designed for use in two-site immunoassays.For example, two bispecific heteromultimers are produced binding to twoseparate epitopes on the analyte protein—one heteromultimer binds thecomplex to an insoluble matrix, the other binds an indicator enzyme.

Heteromultimers can also be used for in vitro or in vivo immunodiagnosisof various diseases such as cancer. To facilitate this diagnostic use,one arm of the heteromultimer can be designed to bind a tumor associatedantigen and the other arm can bind a detectable marker (e.g. a chelatorwhich binds a radionuclide). For example, a heteromultimer havingspecificities for the tumor associated antigen CEA as well as a bivalenthapten can be used for imaging of colorectal and thyroid carcinomas.Other non-therapeutic, diagnostic uses for the heteromultimer will beapparent to the skilled practitioner.

For diagnostic applications, at least one arm of the heteromultimertypically will be labeled directly or indirectly with a detectablemoiety. The detectable moiety can be any one which is capable ofproducing, either directly or indirectly, a detectable signal. Forexample, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, suchas alkaline phosphatase, beta-galactosidase or horseradish peroxidase(HRP).

Any method known in the art for separately conjugating theheteromultimer to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature 144:945 (1962); David et al.,Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

The heteromultimers of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof heteromultimer. The amount of analyte in the test sample is inverselyproportional to the amount of standard that becomes bound to theheteromultimer. To facilitate determining the amount of standard thatbecomes bound, the heteromultimers generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the heteromultimers may conveniently be separated from the standardand analyte which remain unbound.

The heteromultimers are particularly useful for sandwich assays whichinvolve the use of two molecules, each capable of binding to a differentimmunogenic portion, or epitope, of the sample to be detected. In asandwich assay, the test sample analyte is bound by a first arm of theheteromultimer which is immobilized on a solid support, and thereafter asecond arm of the heteromultimer binds to the analyte, thus forming aninsoluble three part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond arm of the heteromultimer may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

EXAMPLES

A strategy is presented for preparing Fc-containing BsAb (FIG. 1C). Inthis strategy, we have engineered the C_(H)3 domain of antibody heavychains so that they heterodimerize but do not homodimerize. This wasaccomplished by installing inter-chain disulfide bonds in the C_(H)3domain in conjunction with sterically complimentary mutations obtainedby rational design (Ridgway et al., supra (1996)) and phage displayselection as described herein. Use of a single light chain for bothantigen binding specificities circumvents the problem of light chainmispairing (FIG. 1A-1C). Antibodies with the same light chain werereadily isolated by panning a very large human scFv library (Vaughan, T.J., et al., (1996) supra).

Example 1 Generation of Protuberance-Into-Cavity HeteromultimerImmunoadhesins

The C_(H)3 interface between the humanized anti-CD3/CD4-IgG chimerapreviously described by Chamow et al. J. Immunol. 153:4268 (1994) wasengineered to maximize the percentage of heteromultimers which could berecovered. Protuberance-into-cavity and wild-type C_(H)3 variants werecompared in their ability to direct the formation of a humanizedantibody-immunoadhesin chimera (Ab/Ia) anti-CD3/CD4-IgG.

Thus, mutations were constructed in the C_(H)3 domain of the humanizedanti-CD3 antibody heavy chain and in CD4-IgG by site-directedmutagenesis using mismatched oligonucleotides (Kunkel et al., MethodsEnzymol. 154:367 (1987) and P. Carter, in Mutagenesis: a PracticalApproach, M. J. McPherson, Ed., IRL Press, Oxford, UK, pp. 1-25 (1991))and verified by dideoxynucleotide sequencing (Sanger et al., Proc. Natl.Acad. Sci. USA 74:5463 (1977)). See Table 3 below.

TABLE 3 C_(H)3 of anti-CD3 C_(H)3 of CD4-IgG Most Preferred MutantsT366Y Y407T T366W Y407A F405A T394W Y407T T366Y T366Y:F405A T394W:Y407TT366W:F405W T394S:Y407A F405W:Y407A T366W:T394S Preferred Mutants F405WT394S

Residue T366 is within hydrogen-bonding distance of residue Y407 on thepartner C_(H)3 domain. Indeed the principal intermolecular contact toresidue T366 is to residue Y407 and vice versa. Oneprotuberance-into-cavity pair was created by inverting these residueswith the reciprocal mutations of T366Y in one C_(H)3 domain and Y407T inthe partner domain thus maintaining the volume of side chains at theinterface. Mutations are denoted by the wild-type residue followed bythe position using the Kabat numbering system (Kabat et al. (1991)supra) and then the replacement residue in single-letter code. Multiplemutations are denoted by listing component single mutations separated bya colon.

Phagemids encoding anti-CD3 light (L) and heavy (H) chain variants(Shalaby et al., J. Exp. Med. 175:217 (1992) and Rodrigues et al., Int.J. Cancer (Suppl.) 7:45 (1992)) were co-transfected into human embryonickidney cells, 293S, together with a CD4-IgG variant encoding phagemid(Byrn et al., Nature 344:667 (1990)) as previously described (Chamow etal., J. Immunol. 153:4268 (1994)). The total amount of transfectedphagemid DNAs was fixed whereas the ratio of different DNAs was variedto maximize the yield of Ab/Ia chimera. The ratio (by mass) of Ia: heavychain:light chain input DNAs (15 μg total) was varied as follows: 8:1:3;7:1:3; 6:1:3; 5:1:3; 4:1:3; 3:1:3; 1:0:0; 0:1:3.

The products were affinity purified using Staphylococcal protein A(ProSep A, BioProcessing Ltd, UK) prior to analysis by SDS-PAGE followedby scanning LASER densitometry. Excess light over heavy chain DNA wasused to avoid the light chain from being limiting. The identity ofproducts was verified by electroblotting on to PVDF membrane(Matsudaira, J. Biol. Chem. 262:10035 (1987)) followed by amino terminalsequencing.

Co-transfection of phagemids for light chain together with those forheavy chain and Ia incorporating wild-type C_(H)3 resulted in a mixtureof Ab/Ia chimera, IgG and Ia homodimer products as expected (Chamow etal., J. Immunol. 153:4268 (1994)). The larger the fraction of input DNAencoding antibody heavy plus light chains or Ia the higher the fractionof corresponding homodimers recovered. An input DNA ratio of 6:1:3 ofIa:H:L yielded 54.5% Ab/Ia chimera with similar fractions of Iahomodimer (22.5%) and IgG (23.0%). These ratios are in good agreementwith those expected from equimolar expression of each chain followed byrandom assortment of heavy chains with no bias being introduced by themethod of analysis: 50% Ab/Ia chimera, 25% Ia homodimer and 25% IgG.

In contrast to chains containing wild-type C_(H)3, Ab/Ia chimera wasrecovered in yields of up to 92% from cotransfections in which theanti-CD3 heavy chain and CD4-IgG Ia contained the Y407T cavity and T366Yprotuberance mutations, respectively. Similar yields ofantibody/immunoadhesin chimera were obtained if these reciprocalmutations were installed with the protuberance on the heavy chain andthe cavity in the Ia. In both cases monomer was observed for the chaincontaining the protuberance but not the cavity. Without being limited toany one theory, it is believed that the T366Y protuberance is moredisruptive to homodimer formation than the Y407T cavity. The fraction ofAb/Ia hybrid was not significantly changed by increasing the size ofboth protuberance and cavity (Ab T366W, Ia Y407A). A second protuberanceand cavity pair (Ab F405A, Ia T394W) yielded up to 71% Ab/Ia chimerausing a small fraction of Ia input DNA to offset the unanticipatedproclivity of the Ia T394W protuberance variant to homodimerize.Combining the two independent protuberance-into-cavity mutant pairs (AbT366Y:F405A, Ia T394W:Y407T) did not improve the yield of Ab/Ia hybridover the Ab T366Y, Ia Y407T pair.

The fraction of Ab/Ia chimera obtained with T366Y and Y407T mutant pairwas virtually independent of the ratio of input DNAs over the rangetested. Furthermore the contaminating species were readily removed fromthe Ab/Ia chimera by ion exchange chromatography (0-300 mM NaCl in 20 mMTris-HCl, pH 8.0) on a mono S HR 5/5 column (Pharmacia, Piscataway,N.J.). This augurs well for the preparation of larger quantities Ab/Iachimeras using stable cell lines where the relative expression levels ofAb and Ia are less readily manipulated than in the transient expressionsystem.

The protuberance-into-cavity mutations identified are anticipated toincrease the potential applications of Fc-containing BsAb by reducingthe complexity of the mixture of products obtained from a possible tenmajor species (Suresh et al., Methods Enzymol. 121:210 (1990)) down tofour or less (FIGS. 1A-1B). It is expected that the T366Y and Y407Tmutant pair will be useful for generating heteromultimers of other humanIgG isotypes (such as IgG₂, IgG₃ or IgG₄) since T366 and Y407 are fullyconserved and other residues at the C_(H)3 domain interface of IgG₁ arehighly conserved.

Example 2 Generation of Non-Naturally Occurring Disulfide Linkages inHeteromultimeric Immunoadhesins

A. Design of C_(H)3 Inter-Chain Disulfide Bonds.

Three criteria were used to identify pairs of residues for engineering adisulfide bond between partner C_(H)3 domains: i) The Cα separationpreferably is similar to those found in natural disulfide bonds (5.0 to6.8 Å) (Srinivasan, N., et al., Int. J. Peptides Protein Res. 36:147-155(1990)). Distances of up to 7.6 Å were permitted to allow for main chainmovement and to take into account the uncertainty of atomic positions inthe low resolution crystal structure (Deisenhofer, Biochemistry20:2361-2370 (1981)). ii) The Cα atoms should be on different residueson the two C_(H)3 domains. iii) The residues are positioned to permitdisulfide bonding (Srinivasan, N., et al., (1990) supra).

B. Modeling of disulfide bonds. Disulfide bonds were modeled into thehuman IgG₁ Fc (Deisenhofer, supra) as described for humAb4D5-Fv(Rodrigues et al., Cancer Res. 55:63-70 (1995)) using Insight II release95.0 (Biosym/MSI).

C. Construction of C_(H)3 variants. Mutations were introduced into theC_(H)3 domain of a humanized anti-CD3 heavy chain or CD4-IgG bysite-directed mutagenesis (Kunkel, et al., Methods Enzymol. 154:367-382(1987)) using the following synthetic oligonucleotides:

Y349C, (SEQ. ID NO: 1) 5′ CTCTTCCCGAGATGGGGGCAGGGTGCACACCTGTGG 3′ S354C,(SEQ. ID NO: 2) 5′ CTCTTCCCGACATGGGGGCAG 3′ E356C, (SEQ. ID NO: 3)5′ GGTCATCTCACACCGGGATGG 3′ E357C, (SEQ. ID NO: 4)5′ CTTGGTCATACATTCACGGGATGG 3′ L351C, (SEQ. ID NO: 5)5′ CTCTTCCCGAGATGGGGGACAGGTGTACAC 3′ D399C, (SEQ. ID NO: 6)5′ GCCGTCGGAACACAGCACGGG 3′ K392C, (SEQ. ID NO: 7)5′ CTGGGAGTCTAGAACGGGAGGCGTGGTACAGTAGTTGTT 3′ T394C, (SEQ. ID NO: 8)5′ GTCGGAGTCTAGAACGGGAGGACAGGTCTTGTA 3′ V397C, (SEQ. ID NO: 9)5′ GTCGGAGTCTAGACAGGGAGG 3′ D399S, (SEQ. ID NO: 10)5′ GCCGTCGGAGCTCAGCACGGG 3′ K392S, (SEQ. ID NO: 11)5′ GGGAGGCGTGGTGCTGTAGTTGTT 3′ C231S: C234S (SEQ. ID NO: 12)5′ GTTCAGGTGCTGGGCTCGGTGGGCTTGTGTGAGTTTTG 3′

Mutations are denoted by the amino acid residue and number (Eu numberingscheme of Kabat et al., supra (1991), followed by the replacement aminoacid. Multiple mutations are represented by the single mutationseparated by a colon. Mutants were verified by dideoxynucleotidesequencing (Sanger et al., supra (1977)) using Sequenase version 2.0(United States Biochemicals, Cleveland, Ohio).

D. An inter-chain disulfide enhances heterodimer formation. Six pairs ofmolecules containing inter-chain disulfide bonds in the C_(H)3 domain(“disulfide-C_(H)3” variants; v1-v6, Table 4) were compared with parentmolecules in their ability to direct the formation of an Ab/Ia hybrid,anti-CD3/CD4-IgG (Chamow et al., supra (1994)). Plasmids encodingCD4-IgG and anti-CD3 heavy chain variants were co-transfected into 293Scells, along with an excess of plasmid encoding the anti-CD3 lightchain. The yield of heterodimer was optimized by transfecting with arange of Ia:H chain:L chain DNA ratios. The Ab/Ia heterodimer, IgG andIa homodimer products were affinity-purified using Staphylococcalprotein A and quantified by SDS-PAGE and scanning laser densitometry(Ridgway et al., supra (1996)).

Each disulfide-C_(H)3 pair gave rise to three major species, similar tothe parent molecules. However, Ab/Ia heterodimer from disulfide-C_(H)3variants was shifted in electrophoretic mobility, consistant withformation of an inter-chain disulfide in the C_(H)3 domain. Furtherevidence of disulfide bond formation was provided by the inter-chaindisulfides in the hinge. Covalently bonded Ab/Ia hybrids were observedby SDS-PAGE for disulfide-C_(H)3 variants but not for molecules withwildtype C_(H)3 domains in which hinge cysteines were mutated to serine.Disulfide-C_(H)3 variants were prepared and designated Y349C/S354° C.,Y349C/E356° C., Y349C/E357° C., L351C/E354° C., T394C/E397° C., andD399C/K392C. Only one variant (D399C/K392° C.) substantially increasedthe yield of Ab/Ia hybrid over wildtype (76% vs. 52%, respectively) asdetermined by SDS-PAGE analysis of the variants. Mutations are denotedby the amino acid residue and number (Eu numbering scheme of Kabat etal. (1991) supra), followed by the replacement amino acid. Mutations inthe first and second copies of C_(H)3 come before and after the slash,respectively. Residues in the second copy of C_(H)3 are designated witha prime (′). This improvement apparently reflects disulfide bondformation rather than replacement of residues K392 and D399, since themutations K392S/D399′S gave both a similar Ab/Ia yield and Ab/Iaelectrophoretic mobility relative to wildtype. Homodimers migratedsimilarly to those with wildtype Fc domains, demonstrating preferentialengineered inter-chain disulfide bond formation in the C_(H)3 domain ofheterodimers. All disulfide-C variants were expressed at approximatelythe same level as the parent molecules in 293S cells.

E. Disulfides combined with protuberance-into-cavity engineeringincreases the yield of heterodimer to 95%. The best disulfide pairincreased the percent of heterodimer to 76% and theprotuberance-into-cavity strategy increased the percent of heterodimerto 87% (Table 4; see also Ridgway et al., (1996) supra). These twostrategies rely on different principles to increase the probability ofgenerating heterodimer. Therefore, we combined the two strategies,anticipating further improvement in the yield of heterodimer. Two of themodeled disulfides, containing L351C or T394C, could potentially formdisulfide-bonded homodimers as well as disulfide-bonded heterodimers(L351C/S354° C. and T394C/V397° C.), thus decreasing their utility. Theremaining four disulfide pairs were installed into the phage-selectedheterodimer (variants v9-v16) and assayed for the yield of heterodimer(Table 4). Yields of approximately 95% heterodimer were obtained. Again,the heterodimer showed an electrophoretic mobility shift compared towildtype and v8 variants.

TABLE 4 Yields of Heterodimers from C_(H)3 Variants Yield of Mutationsheterodimer Variant Subunit A Subunit B (%) wildtype — — 51 ± 1 v1 Y349CS354C 54 ± 4 v2 Y349C E356C 55 ± 6 v3 Y349C E357C 57 ± 4 v4 L351C E354C56 ± 3 v5 T394C E397C 57 ± 2 v6 D399C K392C 73 ± 3 v7 D399S L392S 55 ± 1v8 T366W T366S: L368A: Y407V 86.7 ± 2.3 v9 T366W: D399C T366S: L368A:K392C: Y407V 86.5 ± 0.5 v11 S354C: T366W Y349C: T366S: L368A: Y407V 95 ±2 v12 E356C: T366W Y349C: T366S: L368A: Y407V 94 ± 2 v13 E357C: T366WY349C: T366S: L368A: Y407V 93 ± 2 v14 T366W: K392C T366S: D399C: L368A:Y407V 92 ± 1 v15 Y349C: T366W S354C: T366S: L368A: Y407V 90 ± 1 v16Y349C: T366W E356C: T366S: L368A: Y407V 95.5 ± 0.5 v17 Y349C: T366WE357C: T366S: L368A: Y407V 91.0 ± 1.0

Example 3 Structure-Guided Phage Display Selection for ComplementaryMutations that Enhance Protein-Protein Interaction in Heteromultimers

The following strategy is useful in the selection of complementarymutations in polypeptides that interact at an interface via amultimerization domain. The strategy is illustrated below as it appliesto the selection of complementary protuberance-into-cavity mutations.However, the example is not meant to be limiting and the strategy may besimilarly applied to the selection of mutations appropriate for theformation of non-naturally occurring disulfide bonds, leucine zippermotifs, hydrophobic interactions, hydrophilic interactions, and thelike.

A. Phage display selection. A phage display strategy was developed forthe selection of stable C_(H)3 heterodimers and is diagrammed in FIG. 2.The selection uses a protuberance mutant, T366W (Ridgway et al., supra(1996)), fused to a peptide flag (gD peptide flag, for example, Lasky,L. A. and Dowbenko, D. J. (1984) DNA 3:23-29; and Berman, P. W., et al.(1985) Science 227:1490-1492) that is coexpressed with a second copy ofC_(H)3 fused to M13 gene III protein. A library of cavity mutants wascreated in this second copy of C_(H)3 by randomization of the closestneighboring residues to the protuberance on the first C_(H)3 domain.Phage displaying stable C_(H)3 heterodimers were then captured using ananti-flag Ab.

A C_(H)3 phage display library of 1.1×10⁵ independent clones wasconstructed by replacement of a segment of the natural C_(H)3 gene witha PCR fragment. The fragment was obtained by PCR amplification usingdegenerate primers to randomize positions 366, 368 and 407 usingstandard techniques.

After 2 to 5 rounds of selection, the fraction of full length clones was90%, 60%, 50% and 10%, respectively, as judged by agarose gelelectrophoresis of single-stranded DNA. Phagemids containing full lengthclones were gel-purified after 5 rounds of selection. Two thousandtransformants were obtained after retransforming XL1-BLUE™ cells(Stratagene).

A mean of >10⁶ copies of each clone was used per round of panning. Thus,numerous copies of each clone in the library were likely available forselection, even though some deletion mutants arose during panning.

After 7 rounds of panning, the C_(H)3 mutants obtained approached aconsensus amino acid sequence at the randomized residues. Virtually allclones had serine or threonine at residue 366 indicating a very strongpreference for a β-hydroxyl at this position. A strong preference forhydrophobic residues was observed for residues 368 and 407, with valineand alanine predominating. Six different amino acid combinations wererecovered at least twice, including the triple mutant,T366S:L368A:Y407V, which was recovered 11 times. None of these phageselectants has an identical sequence to a previously designedheterodimer, T366W/Y407′A (Ridgway, J. B. B., et al., (1996), supra. Thephage selectants may be less tightly packed than the wild-type C_(H)3homodimer as judged by a 40-80 Å³ reduction in total side chain volumeof the domain interface residues.

C_(H)3 variants encoded on the expression plasmid pAK19 (Carter et al.1992) were introduced into E. coli strain 33B6, expressed, and secretedfrom E. coli grown to high cell density in a fermentor. TheT366S:L368A:Y407V mutant purified by DEAE-Sepharose FF, ABx and ResourceS chromatography gave a single major band following SDS-PAGE. OtherC_(H)3 variants were recovered with similar purity. The molecular massesof wild-type C_(H)3 and T366S:L368A:Y407V, T366W and Y407A variantsdetermined by high resolution electrospray mass spectrometry were asexpected.

B. Phage-selected heterodimer stability. The stability of C_(H)3heterodimers was first assessed by titrating corresponding phage withguanidine hydrochloride, followed by dilution and quantification ofresidual heterodimer by enzyme-linked immunosorbent assay (ELISA). Theguanidine hydrochloride denaturation assay with C_(H)3-phage provides ameans to screen selectants rapidly.

Phage were prepared from individual clones following 7 rounds ofselection and also from the control vector, pRA1. Briefly, phagemids inXL1-BLUE™ were used to inoculate 25 ml LB broth containing 50 μg/mlcarbenicillin and 10 μg/ml tetracycline in the presence of 10⁹ pfu/mlM13K07 and incubated overnight at 37° C. The cells were pelleted bycentrifugation (6000 g, 10 min, 4° C.). Phage were recovered from thesupernatant by precipitation with 5 ml 20% (w/v) PEG, 2.5 M NaClfollowed by centrifugation (12000 g, 10 min, 4° C.) and then resuspendedin 1 ml PBS. 180 μl 0-6 M guanidine hydrochloride in PBS was added to 20μl phage preparations and incubated for 5.0 min at approximately ˜25° C.Aliquots (20 μl) of each phage sample were then diluted 10-fold withwater. The presence of C_(H)3 heterodimer was assayed by ELISA using5B6-coated plates and detecting the phage with an anti-M13 polyclonal Abconjugated to horseradish peroxidase, using o-phenylenediamine as thesubstrate. The reaction was quenched by the addition of 50 μl 2.5 MH₂SO₄ and the absorbance measured at 492 nm. The absorbance data wereplotted against the guanidine hydrochloride concentration during themelt and fitted to a 4 parameter model by a non-linear least squaresmethod using Kaleidagraph 3.0.5 (Synergy Software).

The most frequently recovered heterodimer, T366W/T366′S:L368′A:Y407′V,is similar in stability to other phage-selected heterodimers. Thisphage-selected heterodimer is significantly more stable than thedesigned heterodimer, T366W/Y407′A but less stable than the wild-typeC_(H)3. All C_(H)3 variants, both individually and in combination, werefound to be dimers by size exclusion chromatography under the conditionsthat these same molecules were studied by calorimetry (1.75 mg/ml, inphosphate-buffered saline (PBS)). The only exception was theT366S:L368A:Y407V mutant alone which had a slightly shorter retentiontime than C_(H)3 dimers.

A 1:1 mixture of T366W, protuberance, and T366S:L368A:Y407V, cavity,mutants melts with a single transition at 69.4° C., consistent withsubunit exchange and formation of a stable heterodimer. In contrast, theT366W protuberance homodimer is much less stable than theT366W/T366′S:L368′A:Y407′V protuberance-into-cavity heterodimer(ΔT_(m)=−15.0° C.). The T366S:L368A:Y407V cavity mutant on its own isprone to aggregate upon heating and does not undergo a smooth meltingtransition.

The designed cavity mutant, Y407A, melts at 58.8° C. and 65.4° C. in theabsence and presence of the T366W protuberance mutant, respectively.This is consistent with subunit exchange and formation of a T366W/Y407′Aheterodimer that has greater stability than either T366W (ΔT_(m)=11.0°C.) or Y407A (ΔT_(m)=6.6° C.) homodimers. The phage-selectedheterodimer, T366W/T366′S:L368′A:Y407′V, is more stable than thedesigned heterodimer, T366W/Y407′A, (ΔT_(m)=4.0° C.), but is less stablethan the wild-type C_(H)3 homodimer (ΔT_(m)=−11.0° C.).

C. Multimerization of a phage-selected antibody immunoadhesin (Ab/Ia) invivo. Phage-selected and designed C_(H)3 mutants were compared in theirability to direct the formation of an Ab/Ia hybrid, anti-CD3/CD4-IgG invivo (Chamow et al., (1994), supra. This was accomplished bycoexpression of humanized anti-CD3 light (L) and heavy chains togetherwith CD4-IgG. Formation of heterodimers and homodimers was assessed byprotein A purification followed by SDS-PAGE and scanning laserdensitometry (Ridgway, et al., (1996), supra). Comparable yields ofAb/Ia hybrid were recovered from cotransfections in which the anti-CD3heavy chain contained the designed protuberance mutation, T366W, and theIa contained either the phage-selected mutations, T366S:L368A:Y407V, ordesigned cavity mutation, Y407A (FIG. 3).

Phage-selected and designed C_(H)3 mutants were next evaluated in theirpropensity to form homodimers. The protuberance mutation, T366W, isapparently very disruptive to homodimerization since cotransfection ofcorresponding antibody heavy and light chains leads to an excess of HLmonomers (may include non disulfide-bonded IgG) over IgG. In contrast,IgG but no HL monomers are observed for the same antibody containingwild-type C_(H)3 domains. The cavity mutations, T366S:L368A:Y407V, aresomewhat disruptive to homodimerization since transfection of thecorresponding phagemid leads to a mixture of predominantly Ia dimerswith some Ia monomers. The cavity mutation, Y407A, is minimallydisruptive to homodimerization as judged by the presence of Ia dimersbut no Ia monomers following transfection of the corresponding phagemid.

The phage display selection strategy described herein allows theselection in favor of C_(H)3 mutants that form stable heterodimers andselection against mutants that form stable homodimers. The counterselection against homodimers occurs because “free” C_(H)3 mutants willcompete with the flagged C_(H)3 knob mutant for binding to availableC_(H)3 mutant-gene III fusion protein. The free C3_(H) mutants arise asa result of the amber mutation between the natural C_(H)3 gene and M13gene III. In an amber suppressor host such as XL1-Blue, both C_(H)3-geneIII fusion protein and corresponding free C_(H)3 will be secreted.

Guanidine hydrochloride denaturation proved to be a useful tool for thepreliminary screening of the stability of C_(H)3 heterodimers on phage.Phage maintain infectivity for E. coli even after exposure to 5 Mguanidine hydrochloride (Figini et al., J. Mol. Biol. 239:68-78 (1994)).Thus, guanidine may also be useful to increase the stringency of mutantselection.

Rational design and screening of phage display libraries arecomplementary approaches to remodeling a domain interface of a homodimerto promote heterodimerization. In the case of C_(H)3 domains, designedmutants identified domain interface residues that could be recruited topromote heterodimerization. Phage display was then used here to searchpermutations of 3 residues neighboring a fixed protuberance forcombinations that most efficiently form heterodimers. Phage selectantsare useful to facilitate further rational redesign of the domaininterface, while the phage selection strategy described hereindemonstrates its usefulness for remodeling protein-protein interfaces.

Example 4 Generation and Assembly of Heteromultimeric Antibodies orAntibody/Immunoadhesins Having Common Light Chains

The following example demonstrates preparation of a heteromultimericbispecific antibody sharing the same light chain according to theinvention and the ability of that antibody to bind its target antigens.

A. Identification of Antibodies that Share the Same Light Chain:Comparison of Antibody Libraries Raised to Eleven Antigens.

A large human single chain Fv (scFv) antibody library (Vaughan et al.(1996), supra) was panned for antibodies specific for eleven antigensincluding Axl(human receptor tyrosine kinase ECD), GCSF-R (humangranulocyte colony stimulating factor receptor ECD), IgE (murine IgE),IgE-R (human IgE receptor α-chain), MPL (human thrombopoietin receptortyrosine kinase ECD), MusK (human muscle specific receptor tyrosinekinase ECD), NpoR (human orphan receptor NpoR ECD), Rse (human receptortyrosine kinase, Rse, ECD), HER3 (human receptor tyrosine kinaseHER3/c-erbB3 ECD), Ob-R (human leptin receptor ECD), and VEGF (humanvascular endothelial growth factor) where ECD refers to theextracellular domain. The nucleotide sequence data for scFv fragmentsfrom populations of antibodies raised to each antigen was translated toderive corresponding protein sequences. The V_(L) sequences were thencompared using the program “align” with the algorithm of Feng andDoolittle (1985, 1987, 1990) to calculate the percentage identitybetween all pairwise combinations of chains (Feng, D. F. and Doolittle,R. F. (1985) J. Mol. Evol. 21:112-123; Feng, D. F. and Doolittle, R. F.(1987) J. Mol. Evol. 25:351-360; and Feng, D. F. and Doolittle, R. F.(1990) Methods Enzymol. 183:375-387). The percent sequence identityresults of each pairwise light chain amino acid sequence comparison werearranged in matrix format (see Appendix).

For most pairwise comparisons, at least one common light chain sequencewas found. Table 5 is a comparison of the V_(L) chains showing thefrequencies of scFv sharing identical light chains (100% identity)determined by alignment of 117 V_(L) amino acid sequences. For example,the entry 4/9 (HER3×Ob-R, highlighted in a black box), denotes that 4clones that bind HER3 were found to share their V_(L) sequence with oneor more anti-Ob-R clones, whereas 9 clones binding the Ob-R share theirV_(L) sequence with one or more anti-HER3 clones. The entries on thediagonal represent the number of antibody clones within a populationthat share a V_(L) sequence with one or more clones in the population.For example, examination of the MPL clones revealed 5 clones that sharedtheir V_(L) sequence with one or more other MPL clones. In the caseswhere no common light chain sequence was observed, such as for (IgE×Axl)or (NpoR×IgE-R), the number of fragments compared for at least onespecificity was very small (5 or less). Given the number of common lightchains found, it is likely that common light chains can be found for anyV_(L) comparison if a sufficient number of clones are compared.

The amino acid sequences of light chains were examined for the positionsof amino acid residue differences when the sequence identity relative toa chosen common light chain was 98% and 99%. FIG. 4 is a comparison ofV_(L) sequences of eight different antibodies with specificities for Axl(clone Axl.78), Rse (clones Rse.23, Rse.04, Rse.20, and Rse.15), IgER(clone IgER.MAT2C1G11), Ob-R (clone obr.4), and VEGF (clone vegf.5). Theposition of the antigen binding CDR residues according to a sequencedefinition (Kabat, G. A., et al. (1991) supra) or structural definition(Chothia and Lesk, (1987) J. Mol. Biol. 196:901-917) are shown byunderlining and #, respectively. Light chain residues that differ fromthe Axl.78 sequence are shown by double underlining. Of the 9 lightchains compared, 6 are identical. The light chains of Rse.04 and obr.4(approximately 99% sequence identity) differ by one residue outside ofthe antigen binding CDRs. The light chain of Rse.20 (approximately 98%sequence identity) differs by two residues outside of the antigenbinding CDRs. The amino acid residue changes may have little or noaffect on antigen binding. Thus, the sequence similarity of these lightchains makes them candidates for the common light chain of theinvention. Alternatively, according to the invention, such light chainshaving 98-99% sequence identity with the light chain of a prospectivepaired scFv (Axl.78, for example) may be substituted with the pairedlight chain and retain binding specificity.

B. Identification of Antibodies that Share the Same Light Chain andConstruction of a Bispecific Antibody Sharing that Light Chain:Anti-Ob-R/Anti-HER3.

ScFv fragments that bound human leptin receptor (Ob-R) or theextracellular domain of the HER3/c-erbB3 gene product (HER3) wereobtained by three rounds of panning using a large human scFv phagelibrary (Vaughan et al. (1996), supra). Leptin receptor-IgG and HER3-IgG(10 μg in 1 ml PBS were used to coat separate Immunotubes (Nunc;Maxisorp) overnight at 4° C. Panning and phage rescue were thenperformed as described by Vaughan et al. (1996), supra, with thefollowing modifications. A humanized antibody, huMAb4D5-8 (Carter, P. etal. (1992) PNAS USA 89:4285-4289) or humanized anti-IgE (Presta, L. etal. (1993) J. Immunol. 151:2623-2632) at a concentration of 1 mg/ml wasincluded in each panning step to absorb Fc-binding phage. In addition,panning in solution (Hawkins, R. E., et al. (1992) J. Mol. Biol.226:889-896) was also used to identify scFv binding leptin receptor. Theleptin receptor was separated from the Fc by site-specific proteolysisof leptin receptor-IgG with the engineered protease, Genenase (Carter,P., et al. (1989) Proteins: Structure, Function and Genetics 6:240-248)followed by protein A Sepharose chromatography. The leptin receptor wasbiotinylated and used at a concentration of 100 nM, 25 nM and 5 nM forthe first, second, and third rounds of panning, respectively. Phagebinding biotinylated antigen were captured using streptavidin-coatedparamagnetic beads (Dynabeads, Dynal, Oslo, Norway).

Clones from rounds 2 and 3 of each panning were screened by phage andscFv ELISA using the corresponding antigen and also a controlimmunoadhesin or antibody. The diversity of antigen-positive clones wasanalyzed by PCR-amplification of the scFv insert using the primers,fdtetseq and PUC reverse (Vaughan et al. (1996), supra) and by digestionwith BstNI (Marks et al. (1991) supra). One to five clones per BstNIfingerprint were then cycle-sequenced using fluorescent dideoxy chainterminators (Applied Biosystems) using PCR heavy link and myc seq 10primers (Vaughan et al. (1996), supra). Samples were analyzed using anApplied Biosystems Automated DNA Sequencer and sequences analyzed usingSeqEd. It is also noted that the quanidine hydrochloride antibodydenaturation and in vitro chain shuffling method of Figini combined withphage display selection is useful as a method of selecting antibodieshaving the same light chain (Figini, M. et al. (1994), supra, hereinincorporated by reference in its entirety).

Using the method described above, eleven different anti-HER3 clones and18 anti-Ob-R clones (11 form panning using coated antigen and 7 frompanning with biotinylated antigen) were obtained. The clones weresequenced by standard techniques to determine the sequences of the lightchains associated with each binding domain (FIG. 5). The sequences arethe V_(H) and common V_(L) sequences of the anti-Ob-R clone 26 andanti-HER3 clone 18 used to construct a bispecific antibody (see below).The residues are numbered according to (Kabat, E. A., et al. (1991)supra). The position of the antigen binding CDR residues according to asequence definition (Kabat et al. (1991) supra) or structural definition(Chothia and Lesk, (1987) J. Mol. Biol. (1987) 196:901-917) are shown byunderlining and overlining, respectively. Identity between residues inthe V_(H) sequences is indicated by *.

The sequences of the light chains were compared for multiple anti-HER3clones relative to multiple anti-Ob-R clones (FIG. 8 and Table 5). Itwas observed that four out of eleven anti-HER3 clones share identicalV_(L) with one or more anti-Ob-R receptor clones. Conversely, nine outof eighteen anti-Ob-R clones share the same V_(L) as one of theanti-HER3 clones (See Table 5, blackened box).

TABLE 5 Shared V_(L) usage by scFv against different target antigensAntigen # Specificity scFv Axl GCSF-R IgE IgE-R MPL MusK NpoR Rse HER3Ob-R VEGF Axl 12 2 2/2 0/0 1/1 2/3 1/1 0/0 3/5 2/2 2/5 1/1 GCSF-R 11 01/1 2/2 2/3 1/1 2/2 2/3 2/2 3/3 2/3 IgE 2 0 1/1 1/1 0/0 1/1 1/1 1/1 1/10/0 IgE-R 4 0 1/1 0/0 1/1 2/3 1/1 1/1 1/1 MPL 23 5 5/3 3/2 5/8

5/9 2/2 MusK 3 0 1/1 1/2 2/2 1/1 1/2 NpoR 5 0 1/1 2/2 2/2 1/2 Rse 20 77/4 5/8 2/1 HER3 11 3

4/4 Ob-R 18 7 1/2 VEGF 8 2

Construction of anti-Ob-R/anti-HER3, a bispecific antibody having acommon light chain was performed as follows. Altered C_(H)3 first andsecond polypeptides having the complementary protuberances and cavitiesas well as the non-naturally occurring disulfide bonds between the firstand second polypeptides were used in the construction of a Fc-containingbispecific antibody. The V_(L) from anti-Ob-R clone #26 and anti-HER3clone #18, which clones share the same light chain, as well as the heavychains from each antibody were used to prepare the bispecific antibodyaccording to the procedures disclosed herein.

This antibody had an electrophoretic mobility shift in apparentmolecular weight relative to a bispecific antibody that differed only bya lack of alterations for generating non-natural disulfide bonds. An 8%SDS-PAGE gel of heterodimeric antibody variants with and withoutnon-naturally occurring disulfide bonds showed a mobility shift fromapproximately 230 apparent MW for wild type heterodimer to approximately200 apparent MW for a heterodimer having one non-natural disulfide bond.The MW shift was sufficient to allow determination of the percent ofeach variant that successfully formed the non-natural disulfide bond.

The binding specificity for both Ob-R and for HER3 of the bispecificantibody is tested by standard ELISA procedures such as the followingmethod. Ob-R binding is demonstrated in an ELISA assay with Ob-R presentas an Ob-R-Ig fusion protein. The Ob-R-Ig fusion protein is coated ontothe well of a 96-well microtitre plate and the bispecific antibody isadded.

The well is washed several times to eliminate non-specific binding toOb-R-Ig. As a second component in the same assay, a biotinylated HER3-Igfusion protein is added and detected by means ofstreptavidin-horseradish peroxidase complex binding to the biotinylatedHER3-Ig fusion protein. Binding is detected by generation of a colorchange upon addition of hydrogen peroxide and TMB peroxidase substrate(Kirkegaard and Perry Laboratories, Gaithersburg, Md.).

Under the conditions just described, the binding of a bispecificantibody to both Ob-R-Ig and to HER3-Ig would be observed as detectablelabel immobilized on the surface of the microtitre well due to theformation of a complex comprising immobilized Ob-R-Ig/bispecificantibody/HER3-Ig biotin/detectably labeled streptavidin. Antibodies thatbind Ob-R-Ig, but not HER3-Ig, do not form the above complex, providinga negative result. Similarly, antibodies that bind to HER3-Ig, but notOb-R-Ig, do not form the above complex and provide a negative result. Incontrast, the bispecific antibody expected to bind both Ob-R-Ig andHER3-Ig, forms the complex yielding a positive result in the assay,demonstrating that the bispecific antibody, having a common light chain,binds both HER3 and Ob-R.

Expression and purification of the anti-(Ob-R/HER3) bispecific antibodywas performed as follows. Human embryonic kidney 293S cells weretransfected with three plasmid DNAs each separately encoding anti-Ob-Rheavy chain, anti-HER3 heavy chain, or the light chain from clone 26 or18 that was common to each of the antibodies, as described supra. Foreach transfection, the ratio of heavy chain-encoding DNA to lightchain-encoding DNA was 1:3 so that light chain would not be limiting forassembly of anti-Ob-R/anti-HER3 bispecific antibody. Both heavy chainswere transfected in a 1:1 ratio with respect to each other. 12 μg oftotal plasmid DNA was then co-transfected into 293S cells by means ofcalcium phosphate precipitation (Gorman, C., DNA Cloning, Vol. II, D. M.Glover, ed., IRL Press, Oxford, p. 143 (1985)). The cells were washedwith PBS prior to adding growth media intended to enhance proteinexpression. Fc-containing proteins were purified from cell supernatantsusing immobilized protein A (ProSep A, BioProcessing Ltd., UK) andbuffer-exchanged into PBS. Iodoacetamide was added to proteinpreparations to a final concentration of 50 mM to prevent reshuffling ofdisulfide bonds.

As an additional example, expression and purification of ananti-(CD3/CD4) antibody/immunoadhesin was performed as follows. Humanembryonic kidney 293S cells were transfected with three plasmid DNAs,each plasmid separately encoding anti-CD3 light chain, anti-CD3 IgG₁heavy chain, or anti-CD4 IgG₁ immunoadhesin. For each transfection, theratio of light chain-encoding DNA to heavy chain-encoding DNA was 3:1 sothat light-chain would not be limiting for assembly of anti-CD3 IgG.Additionally, because the immunoadhesin is poorly expressed, the ratioof immunoadhesin encoding plasmid was added in excess to heavy chainencoding plasmid. The ratios tested ranged from 3:1:3 through 8:1:3 forimmunoadhesin:heavy chain:light chain phagemids. 10 μg total plasmid DNAwere then co-transfected into 293S cells by means of calcium phosphateprecipitation (Gorman, C. (1985), supra), washing cells with PBS priorto transfection. Fc-containing proteins were purified from cellsupernatants using immobilized protein A (ProSep A, BioProcessing Ltd.,UK) and buffer-exchanged into PBS. Iodoacetamide was added to proteinpreparations to a final concentration of 50 mM to prevent reshuffling ofdisulfide bonds.

In each of the above preparations, protein samples were electrophoresedon 8% polyacrylamide gels (Novex) and visualized by staining with Servablue. Gels were de-stained leaving a faint background in an effort toallow visualization and quantitation of minor contaminants. Dried gelswere scanned with the scanning densitometer (GS-670, BioRad) and proteinproducts were quantitated with Molecular Analyst software.

Non-natural (engineered) disulfide bonds introduced into the C_(H)3domain has been disclosed herein to enhance heterodimer formation. Onepair of polypeptides, K392C/D399° C., enhanced heterodimer formation bygenerating up to 76% heterodimer (Table 4, variant v6). Moreover, whenthe presence of an inter-chain disulfide bond was combined with theprotuberance-into-cavity technology, approximately 95% heterodimer wasobtained (Table 4 variants v11, v12, and v16). Thus, the method of theinvention of increasing specific protein/protein interaction between thefirst and second polypeptides of a bispecific antibody increases theyield of desired heteromultimer and minimizes the formation of undesiredheteromultimers or homomultimers.

In addition, the method of characterizing the product heteromultimers byelectrophoretic mobility analysis allows for the determination of therelative amount of desired heteromultimers relative to undesiredproducts.

Selection of a common light chain as described herein further increasesyield of the desired heteromultimer by eliminating the possibility ofmispairing between variable heavy chains and light chains of amultispecific antibody.

C. Identification of Antibodies that Share the Same Light Chain andConstruction of a Bispecific Antibody Sharing that Light Chain:Anti-Mpl/Anti-HER3.

Identification, construction and expression of another bispecificantibody of the invention is demonstrated herein. The methods describedin Parts A and B of this example were utilized for the preparation ofthe anti-Mpl/anti-HER3 bispecific antibody.

Using the methods described in Section A of this example (Comparison ofantibody libraries raised to eleven antigens), supra, the V_(H) andV_(L) amino acid sequences of the anti-HER3 scfv were compared with 23scFv that bind to the human thrombopoietin receptor, c-Mpl. Five of theeleven anti-HER3 clones share an identical V_(L) amino acid sequencewith one or more Mpl-binding clones. Conversely, seven out oftwenty-three anti-Mpl scFv shared the same V_(L) as one of the anti-HER3clones (see Table 5, supra, open box). In contrast, the V_(H) amino acidsequences were much more diverse, with an identity level of 40 to 90%between any anti-Mpl and anti-HER3 clone.

The anti-Mpl scFv, 12B5 (Genbank accession number AF048775; SEQ IDNO:27, disclosed in U.S. application Ser. No. 08/918,148, herebyincorporated by reference in its entirety) and anti HER3 scFv clone H6(Genbank accession number AF048774; SEQ ID NO:28) utilize identicalV_(L) sequences and substantially different V_(H) sequences. These scFvfragments were used to construct the anti-Mpl/anti-HER3 bispecific IgGantibody capable of efficient heterodimerization due to the shared lightchain as well as through the use of knobs-into-holes mutations(described herein) and an engineered disulfide bond between the C_(H)3domains. Antibodies that share the same L chain were chosen tocircumvent the problem of L chains pairing with non-cognate H chains.Two naturally occurring hinge region disulfide bonds were also present.The common L chain was cotransfected with the two H chains containingthe C_(H)3 mutations from variant v11. The IgG products were purified byprotein A affinity chromatography and analyzed by SDS-PAGE usingstandard techniques.

The bispecific IgG antibody (BsIgG) preparation gave rise to a singlemajor band showing greater mobility than IgG containing wild-type C_(H)3domains. This increase in electrophoretic mobility was consistent withthe formation of the engineered disulfide bond in the BsIgG forming amore compact protein species.

The ability of the engineered anti-Mpl/anti-HER3 BsIgG antibody to bindboth Mpl and HER3 ECD antigens was assessed using an ELISA as follows.Using PBS buffer in all steps, individual wells of a 96 well plate(Maxisorp, Nunc) were coated overnight with HER3-IgG or Mpl-IgG at 5μg/ml, washed and then blocked for 1 hour with 0.5% (w/v) BSA. Theprimary antibodies were the anti-Mpl×anti-HER3 BsIgG containing themutations, Y349C:T366S:L368A:Y407V/T366′W:S354° C., and correspondingparental anti-Mpl or anti-HER3 IgG with mutated Fc regions. The primaryantibodies (1 μg/mL) were individually incubated at 2 h at 23° C. withbiotinylated HER3-IgG and a 1:5000 dilution of streptavidin-horse radishperoxidase conjugate (Boehringer Mannheim) and then added to the wellsand incubated for an additional 1 h at 23° C. Peroxidase activity wasdetected with TMB reagents as directed by the vendor (Kirkegaard andPerry Laboratories, Inc., Gaithersburg, Md.).

As anticipated, the anti-Mpl/anti-HER3 BsIgG bound efficiently andsimultaneously to each Mpl and HER3 ECD antigens individually as well asto both antigens simultaneously. By contrast, the parental anti-Mpl andparental anti-HER3 IgG bound only to their corresponding cognate antigen(FIG. 6).

D. Antibodies Containing an Engineered Fc Region are Capable ofEfficient Antibody-Dependent Cell-Mediated Cytotoxicity.

To demonstrate that the engineered F_(c) region (C_(H)3 mutations,supra) utilized in generating the exemplified bispecific antibodies ofthe invention is capable of efficient antibody-dependent cell-mediatedcytotoxicity (ADCC), the following experiment was performed.

The C_(H)3 mutations maintain the ability to support efficientantibody-dependent cell-mediated cytotoxicity (ADCC) as demonstratedusing the method of Lewis, G. D. et al. (Lewis, G. D, et al. (1993)Cancer Immunol. Immunother. 37:255-263, hereby incorporated by referencein its entirety). Briefly, cytotoxicity assays were performed with⁵¹Cr-labeled SK-BR-3 and HBL-100 target cells (ATCC accession numbersHTB-30 and 45509, respectively) and human peripheral blood lymphocytesas effector cells. However, unlike Lewis et al., the lymphocytes werenot activated with IL-2.

The C_(H)3 mutations S354:T366W and Y349:T366S:L368A:Y407V wereintroduced separately into the H chain of the humanized anti-HER2antibody, huMAb4D5-5 prepared by Carter et al. (Carter, P. et al. (1992)PNAS USA 89:4285-4289). Antibodies containing remodeled and wild-type Fcregions had similar potency in ADCC with the HER2-overexpressing breastcancer cell line, SK-BR-3 (FIG. 7). Both remodeled and wild-typeantibodies showed comparable, low activity against the normal breastepithelial cell line. The effects in the H-chain are independent of thebinding domains, predicting that these BsIgG's will function inantibody-dependent cell-mediated cytotoxicity.

The instant invention is shown and described herein in what isconsidered to be the most practical, and the preferred embodiments. Itis recognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure. Allreferences provided herein are herein incorporated by reference in theirentirety.

The invention claimed is:
 1. A method of measuring the formation of aheteromeric multispecific antibody from a mixture of polypeptides,wherein the heteromeric multispecific antibody comprises fourpolypeptides, wherein the first polypeptide and the second polypeptideof the four polypeptides each comprise a heavy chain constant domain anda heavy chain variable domain, and the third polypeptide and the fourthpolypeptide of the four polypeptides are each common light chains thatare identical to each other; wherein the first polypeptide and the thirdpolypeptide form a binding domain that binds a first antigen, andwherein the second polypeptide and the fourth polypeptide form a bindingdomain that binds a different antigen; wherein the first polypeptide andthe second polypeptide each further comprise a multimerization domain,wherein the multimerization domain of either the first polypeptide orthe second polypeptide, or of both the first polypeptide and the secondpolypeptide, is altered by amino-acid substitution to form anon-naturally occurring disulfide bond between a free thiol-containingresidue in the multimerization domain of the first polypeptide and afree thiol-containing residue in the multimerization domain of thesecond polypeptide; wherein the first and second polypeptides dimerizeby interaction of the first and second multimerization domains to formthe heteromeric multispecific antibody; the method comprising the stepsof: (i) causing the mixture of polypeptides from which the heteromericmultispecific antibody is formed to migrate in a gel matrix, and (ii)determining the relative amount of a migrating band corresponding to theheteromeric multispecific antibody having a non-naturally occurringdisulfide bond between the first and second polypeptides, and a slowermigrating band corresponding to a heteromultimer lacking a non-naturallyoccurring disulfide bond between the first and second polypeptide. 2.The method of claim 1, wherein the multimerization domains furthercomprise a protuberance-into-cavity interaction between the first andsecond polypeptides.
 3. The method of claim 2, wherein the methodfurther comprises before step (i): expressing from a first nucleic acidthe first polypeptide, wherein the first nucleic acid is altered toencode an amino acid residue having a larger side chain volume, therebygenerating a protuberance on the first polypeptide multimerizationdomain, and expressing from a second nucleic acid the secondpolypeptide, wherein the second nucleic acid is altered to encode anamino acid residue having a smaller side chain volume, therebygenerating a cavity on the second polypeptide multimerization domainwherein the protuberance is positioned to interact with the cavity. 4.The method of claim 3, wherein the step of altering the first or thesecond nucleic acid to generate a protuberance or generating a cavity,or both, occurs by phage display selection.
 5. The method of claim 3,wherein the amino acid residue having a larger side chain volume thanthe substituted amino acid is selected from the group consisting ofarginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W),isoleucine (I) and leucine (L).
 6. The method of claim 3 wherein theamino acid residue having a smaller side chain volume than thesubstituted amino acid is selected from the group consisting of glycine(G), alanine (A), serine (S), threonine (T), and valine (V), and whereinthe import residue is not cysteine (C).
 7. The method of claim 1,wherein the multimerization domain is at least a part of a C_(H)3 domainregion of an antibody constant domain, and the non-naturally occurringdisulfide bond is between the C_(H)3 multimerization domains of thefirst and second polypeptides.
 8. The method of claim 1, wherein theheavy chain constant domain is the heavy chain constant domain of anIgG.
 9. The method of claim 8 wherein the IgG is a human IgG.
 10. Themethod of claim 1, wherein the heavy chain constant domain is a C_(H)3domain.
 11. The method of claim 10 wherein the C_(H)3 domain is a humanC_(H)3 domain.